Method of producing microbial oil containing fatty acids obtained from stramenopile

ABSTRACT

A method for producing a microbial oil includes the steps of: genetically modifying a labyrinthulid by disrupting and/or silencing a gene, or by transforming another gene in addition to the disruption and/or gene silencing of the gene; culturing the labyrinthulid, such that a fatty acid composition accumulated in the labyrinthulid comprises an increased EPA content; and collecting the microbial oil having the increased EPA content from the labyrinthulid. The increased EPA content is not less than 3.3% of a total fatty acid composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending application Ser. No.16/208,047, filed Dec. 3, 2018, which is a Divisional of copendingapplication Ser. No. 14/711,075, filed on May 13, 2015, which is aDivisional of application Ser. No. 13/877,225, filed on Aug. 1, 2013,and currently issued under U.S. Pat. No. 9,062,315, which is a 371 ofPCT International Application No. PCT/JP2011/072650 filed on Sep. 30,2011, which is based upon and claims the benefit of priority of theprior Japanese Patent Application No. 2011-179194, filed on Aug. 18,2011, and Japanese Patent Application No. 2010-224225, filed on Oct. 1,2010. The entire contents of each of the above documents are herebyincorporated by reference into the present application.

The sequence listing was submitted in the present application Ser. No.17/526,464 in a computer readable form under the name of“P23609US03_replacement_sequence_listing.txt” and is hereby incorporatedby reference into the present application. The electronic copy of thesequence listing in the computer readable form, the file size of whichis 242 K bytes, was created on Jan. 29, 2016. The sequence listingsubmitted was also submitted in copending application Ser. No.14/711,075 in a computer readable form under the name of“130412-revised_sequence_listing.txt” and is hereby incorporated byreference into the present application. The electronic copy of thesequence listing in the computer readable form, the file size of whichis 242 K bytes, was created on Jan. 29, 2016.

TECHNICAL FIELD

The present invention relates to a method for transforming stramenopilewhereby genes of stramenopile are disrupted and/or expression thereof isinhibited by genetic engineering. Particularly, the invention relates toa transformation method for disrupting genes associated with fatty acidbiosynthesis and/or inhibiting expression thereof, a method formodifying the fatty acid composition of a stramenopile, a method forhighly accumulating fatty acids in a stramenopile, a stramenopile havingan enhanced unsaturated fatty acid content, a method for producingunsaturated fatty acid from the unsaturated fatty acid content-enhancedstramenopile, a microbial oil comprising the fatty acid obtained frommicroorganisms belonging to stramenopile, especially from the classLabyrinthulomycetes, and a method of producing the microbial oil fromthe microorganisms, among others.

BACKGROUND ART

Polyunsaturated fatty acids (PUFA) represent an important component ofanimal and human nutrition. ω3 polyunsaturated fatty acids (also calledn-3 polyunsaturated fatty acids) such as eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) have a wide range of roles in many aspects ofhealth, including brain development in children, eye functions,syntheses of hormones and other signaling substances, and prevention ofcardiovascular disease, cancer, and diabetes mellitus (Non-PatentDocuments 1 and 2). These fatty acids therefore represent an importantcomponent of human nutrition. Accordingly, there is a need forpolyunsaturated fatty acid production.

Meanwhile, microorganisms of the class Labyrinthulomycetes are known toproduce polyunsaturated fatty acids. Concerning microorganisms of thefamily Thraustochytrium, there are reports of, for example, apolyunsaturated fatty acid-containing phospholipid producing methodusing Schizochytrium microorganisms (Patent Document 1), andThraustochytrium microorganisms having a docosahexaenoic acid producingability (Patent Document 2). For enhancement of food and/or feed by theunsaturated fatty acids, there is a strong demand for a simpleeconomical process for producing these unsaturated fatty acids,particularly in the eukaryotic system.

With regard to the class Labyrinthulomycetes, there have been reportedforeign gene introducing methods for specific strains of the genusSchizochytrium (the genus Auranthiochytrium (Non-Patent Document 4) inthe current classification scheme (Non-Patent Document 3)) (PatentDocuments 3 and 4). Further, a method that causes a change in fatty acidcomposition by means of transformation is known in which a polyketidesynthase (PKS) gene is destroyed to change the resulting fatty acidcomposition (Non-Patent Document 5). However, there is no reportdirected to changing a fatty acid composition by manipulating theenzymes of the elongase/desaturase pathway. Under these circumstances,the present inventors found ways to change fatty acid compositionsthrough introduction of elongase/desaturase genes into various speciesof Labyrinthulomycetes, and have filed a patent application therefor(Patent Document 5).

CITATION LIST Patent Documents

-   Patent Document 1: JP-A-2007-143479-   Patent Document 2: JP-A-2005-102680-   Patent Document 3: JP-A-2006-304685-   Patent Document 4: JP-A-2006-304686-   Patent Document 5: WO2011/037207-   Patent Document 6: WO1997/011094-   Patent Document 7: US Patent Application US2005/0014231-   Patent Document 8: JP-T-2007-532104 (the term “JP-T” as used herein    means a published Japanese translation of a PCT patent application)

Non-Patent Documents

-   Non-Patent Document 1: Poulos A., Lipids, 30, 1-14(1995)-   Non-Patent Document 2: Horrocks L. A. and Yeo Y. K., Pharmacol Res.,    40, 211-225 (1999)-   Non-Patent Document 3: Yokoyama R., Honda D., Mycoscience, 48,    199-211 (2007)-   Non-Patent Document 4: Lecture Summary for the 60th Conference of    The Society for Biotechnology, Japan, p136 (2008)-   Non-Patent Document 5: Lippmeier J. C. et al., Lipids, 44(7),    621-630 (2009)-   Non-Patent Document 6: Tonon T. et al., FEBS Lett., 553, 440-444    (2003).-   Non-Patent Document 7: Thompson J. D. et al., Nucleic Acids Res.,    22, 4673-4680 (1994)-   Non-Patent Document 8: Yazawa K., Lipids, 31, Supple. 297-300 (1996)-   Non-Patent Document 9: Jiang X. et al., Wei Sheng Wu Xue Bao.,    48(2), 176-183 (2008)-   Non-Patent Document 10: PEREIRA S. L. et al., Biochem. J., 378,    665-671 (2004)-   Non-Patent Document 11: Prasher D. C. et al., Gene, 111(2), 229-233    (1992)-   Non-Patent Document 12: Chalfie M. et al., Science, 263, 802-805    (1994)

Non-Patent Document 13: Southern P. J., and Berg, P., J. Molec. Appl.Gen., 1, 327-339 (1982)

-   Non-Patent Document 14: Saitou N. et al., Mol. Biol. Evol., 4,    406-425 (1987)-   Non-Patent Document 15: Ausubel F. M. et al., Current Protocols in    Molecular Biology, Unit 13 (1994)-   Non-Patent Document 16: Guthrie C., Fink G. et al., Methods in    Enzymology: Guide to Yeast Genetics and Molecular Biology, Volume    194 (1991)-   Non-Patent Document 17: Abe E., et al., J. Biochem, 142, 31561-31566    (2006)-   Non-Patent Document 18: Bio-Experiment Illustrated 2, Fundamentals    of Gene Analysis, p117-128, Shujunsha, 1995-   Non-Patent Document 19: Japan Society for Bioscience, Biotechnology,    and Agrochemistry, 77, 2, 150-153 (2003)-   Non-Patent Document 20: Bio-Experiment Illustrated 2, Fundamentals    of Gene Analysis, p63-68, Shujunsha, 1995-   Non-Patent Document 21: Sanger, F. et al., Proc. Natl. Acad. Sci,    74, 5463 (1977)-   Non-Patent Document 22: Meyer, A., et al. J. Lipid Res., 45,    1899-1909 (2004)-   Non-Patent Document 23: Cigan and Donahue, 1987; Romanos et al.,    1992-   Non-Patent Document 24: Qiu, X., et al. J. Biol. Chem., 276, 31561-6    (2001)-   Non-Patent Document 25: DIG Application Manual [Japanese version]    8th, Roche Applied Science

SUMMARY OF THE INVENTION Problems that the Invention is to Solve

The present invention is directed to improving the ability of astramenopile to produce useful substances by way of transformationthrough disruption of stramenopile genes and/or inhibition of expressionthereof by genetic engineering. By modifying the ability to produceuseful substances through disruption of stramenopile genes associatedwith production of useful substances and/or inhibition of expressionthereof by genetic engineering, the invention provides a modificationmethod of a fatty acid composition produced by a stramenopile, a methodfor highly accumulating fatty acids in a stramenopile, an unsaturatedfatty acid producing method, a stramenopile having an enhancedunsaturated fatty acid content, and production of unsaturated fatty acidfrom the unsaturated fatty acid content-enhanced stramenopile. With themodification of a fatty acid composition produced by a stramenopile, andthe method for highly accumulating fatty acids in a stramenopile, thepresent invention enables more efficient production of polyunsaturatedfatty acids.

Means for Solving the Problems

The present inventors conducted intensive studies under the foregoingcircumstances of the conventional techniques, and succeeded intransforming a stramenopile by way of disrupting stramenopile genesand/or inhibiting expression thereof by genetic engineering to greatlyimprove the ability of the stramenopile to produce an unsaturated fattyacid. The present inventors also found a method for modifying the fattyacid composition produced by a stramenopile through disruption ofstramenopile genes or inhibition of expression thereof by geneticengineering, and a method for highly accumulating unsaturated fattyacids in the transformed stramenopile. The present invention wascompleted after further studies and development for practicalapplications.

The gist of the present invention includes the following stramenopiletransformation methods (1) to (12).

-   -   (1) A method for transforming stramenopile, the method including        disrupting a stramenopile gene and/or inhibiting expression        thereof by genetic engineering. In one embodiment, a method for        producing a microbial oil includes the steps of: genetically        modifying a labyrinthulid by disrupting and/or silencing a gene,        or by transforming another gene in addition to the disruption        and/or gene silencing of the gene; culturing the labyrinthulid,        such that a fatty acid composition accumulated in the        labyrinthulid comprises an increased EPA content; and collecting        the microbial oil having the increased EPA content from the        labyrinthulid. The increased EPA content is not less than 3.3%        of a total fatty acid composition.

(2) The method according to (1), wherein the stramenopile belongs to theclass Labyrinthulomycetes.

(3) The method according to (2), wherein the Labyrinthulomycetes aremicroorganisms belonging to the genus Labyrinthula, Althornia,Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium,Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium,Botryochytrium, Parietichytrium, or Sicyoidochytrium.

(4) The method according to (3), wherein the microorganisms areThraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytriumroseum, Parietichytrium sp., or Schizochytrium sp.

(5) The method according to (4), wherein the microorganisms areThraustochytrium aureum ATCC 34304, Parietichytrium sarkarianum SEK 364(FERN BP-11298), Thraustochytrium roseum ATCC 28210, Parietichytrium sp.SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), orSchizochytrium sp. TY12Ab (FERM BP-11421).

(6) The method according to any one of (1) to (5), wherein thestramenopile gene is a gene associated with fatty acid biosynthesis.

(7) The method according to (6), wherein the gene associated with fattyacid biosynthesis is a gene associated with polyketide synthase, fattyacid chain elongase, and/or fatty acid desaturase.

(8) The method according to (7), wherein the fatty acid chain elongaseis a C20 elongase.

(9) The method according to (7), wherein the fatty acid desaturase is a412 desaturase.

(10) The method according to any one of (1) to (9), wherein the methodused to disrupt the stramenopile gene by genetic engineering iselectroporation or a gene-gun technique introducing a loss-of-functiongene or a DNA fragment from which a coding region of the gene isdeleted.

(11) The method according to any one of (1) to (10), wherein the methodused to inhibit expression of the stramenopile gene by geneticengineering is an antisense technique or RNA interference.

(12) The method according to any one of (1) to (11), further includingintroducing a gene associated with fatty acid desaturase.

(13) The method according to (12), wherein the gene associated withfatty acid desaturase is an ω3 desaturase.

Further, the gist of the present invention includes the followingmethods (14) to (26) for modifying the fatty acid composition of astramenopile.

(14) A method for modifying the fatty acid composition of astramenopile, the method including disrupting a stramenopile gene and/orinhibiting expression thereof by genetic engineering.

(15) The method according to (14), wherein the stramenopile belongs tothe class Labyrinthulomycetes.

(16) The method according to (15), wherein the Labyrinthulomycetes aremicroorganisms belonging to the genus Labyrinthula, Althornia,Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium,Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium,Botryochytrium, Parietichytrium, or Sicyoidochytrium.

(17) The method according to (16), wherein the microorganisms areThraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytriumroseum, Parietichytrium sp., or Schizochytrium sp.

(18) The method according to (17), wherein the microorganisms areThraustochytrium aureum ATCC 34304, Parietichytrium sarkarianum SEK 364(FERN BP-11298), Thraustochytrium roseum. ATCC 28210, Parietichytriumsp. SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406),or Schizochytrium sp. TY12Ab (FERM BP-11421).

(19) The method according to any one of (14) to (18), wherein thestramenopile gene is a gene associated with fatty acid biosynthesis.

(20) The method according to (19), wherein the gene associated withfatty acid biosynthesis is a gene associated with polyketide synthase,fatty acid chain elongase, and/or fatty acid desaturase.

(21) The method according to (20), wherein the fatty acid chain elongaseis a C20 elongase.

(22) The method according to (21), wherein the fatty acid desaturase isa 412 desaturase.

(23) The method according to any one of (14) to (22), wherein the methodused to disrupt the stramenopile gene by genetic engineering iselectroporation or a gene-gun technique introducing a loss-of-functiongene or a DNA fragment from which a coding region of the gene isdeleted.

(24) The method according to any one of (14) to (23), wherein the methodused to inhibit expression of the stramenopile gene by geneticengineering is an antisense technique or RNA interference.

(25) The method according to any one of (14) to (24), further includingintroducing a gene associated with fatty acid desaturase.

(26) The method according to (25), wherein the gene associated withfatty acid desaturase is an ω3 desaturase.

Further, the gist of the present invention includes the followingmethods (27) to (29) for highly accumulating fatty acids in astramenopile.

(27) A method for highly accumulating a fatty acid in a stramenopile,wherein the method uses the method of any one of (14) to (26).

(28) The method according to (27), wherein the fatty acid is anunsaturated fatty acid.

(29) The method according to (28), wherein the unsaturated fatty acid isan unsaturated fatty acid of 18 to 22 carbon atoms.

Further, the gist of the present invention includes the following fattyacid (30).

(30) A fatty acid obtained from the stramenopile in which the fatty acidis highly accumulated by using the method of any one of (27) to (29).

Further, the gist of the present invention includes the followingtransformed stramenopiles (31) to (43).

(31) A stramenopile transformed for the modification of the fatty acidcomposition through disruption of its gene and/or inhibition ofexpression thereof by genetic engineering. In one embodiment, alabyrinthulid that has been genetically modified by disrupting and/orsilencing a gene, or by transforming another gene in addition to thedisruption and/or gene silencing of the gene such that a fatty acidcomposition accumulated in the labyrinthulid comprises an increased EPAcontent. The increased EPA content is not less than 3.3% of a totalfatty acid composition.

(32) The stramenopile according to (31), wherein the stramenopilebelongs to the class Labyrinthulomycetes.

(33) The stramenopile according to (32), wherein the Labyrinthulomycetesare microorganisms belonging to the genus Labyrinthula, Althornia,Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium,Aurantiochytrium, Thraustochytrium, Ulkenia, Oblongichytrium,Botryochytrium, Parietichytrium, or Sicyoidochytrium.

(34) The stramenopile according to (33), wherein the microorganisms areThraustochytrium aureum, Parietichytrium sarkarianum, Thraustochytriumroseum, Parietichytrium sp., or Schizochytrium sp.

(35) The stramenopile according to (34), wherein the microorganisms areThraustochytrium aureum ATCC 34304, Parietichytrium sarkarianum SEK 364(FERM BP-11298), Thraustochytrium roseum ATCC 28210, Parietichytrium sp.SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), orSchizochytrium sp. TY12Ab (FERM BP-11421).

(36) The stramenopile according to any one of (31) to (35), wherein thestramenopile gene is a gene associated with fatty acid biosynthesis.

(37) The stramenopile according to (36), wherein the gene associatedwith fatty acid biosynthesis is a gene associated with polyketidesynthase, fatty acid chain elongase, and/or fatty acid desaturase.

(38) The stramenopile according to (36), wherein the fatty acid chainelongase is a C20 elongase.

(39) The stramenopile according to (37), wherein the fatty aciddesaturase is a 412 desaturase.

(40) The stramenopile according to any one of (31) to (39), wherein themethod used to disrupt the stramenopile gene by genetic engineering iselectroporation or a gene-gun technique introducing a loss-of-functiongene or a DNA fragment from which a coding region of the gene isdeleted.

(41) The stramenopile according to any one of (31) to (40), wherein themethod used to inhibit expression of the stramenopile gene by geneticengineering is an antisense technique or RNA interference.

(42) The stramenopile according to any one of (31) to (41), furthercomprising introducing a gene associated with fatty acid desaturase isintroduced.

(43) The stramenopile according to (42), wherein the gene associatedwith fatty acid desaturase is an ω3 desaturase.

Advantage of the Invention

The present invention improves the ability of a stramenopile to produceuseful substances by way of transformation through disruption ofstramenopile genes and/or inhibition of expression thereof by geneticengineering. By modifying the stramenopiles' ability to produce usefulsubstances through disruption of stramenopile genes associated withproduction of useful substances and/or inhibition of expression thereofby genetic engineering, the invention provides a modification method ofa fatty acid composition produced by a stramenopile, a method for highlyaccumulating fatty acids in a stramenopile, an unsaturated fatty acidproducing method, a stramenopile having an enhanced unsaturated fattyacid content, and production of unsaturated fatty acid from theunsaturated fatty acid content-enhanced stramenopile. With themodification of the fatty acid composition produced by a stramenopile,and the method for highly accumulating fatty acids in a stramenopile,the present invention enables more efficient production ofpolyunsaturated fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the result of RACE performed to amplify a T. aureumATCC 34304-derived elongase gene in Example 2-2. [Brief Description ofReference Numerals] 1: 5′-RACE using a synthetic adapter-specificoligonucleotide and a denatured oligonucleotide elo-R; 2: 3′-RACE usinga synthetic adapter-specific oligonucleotide and a denaturedoligonucleotide elo-F; 3: 5′-RACE using only elo-R (negative control);4: 3′-RACE using only elo-F (negative control); 5: 5′-RACE using only asynthetic adapter-specific oligonucleotide (negative control); 6:3′-RACE using only a synthetic adapter-specific oligonucleotide(negative control).

FIG. 2 represents a molecular phylogenetic tree of T. aureum ATCC34304-derived Δ6/Δ9 elongase and Δ5/Δ6 elongase (TaELO1 and TaELO2) ofExample 2-3.

FIG. 3 represents the evaluation of transfectants with the introducedKONeor in Example 2-8. (A), an oligonucleotide primer set used for theevaluation of the transfectants by a PCR performed with template genomicDNA. [Brief Description of Reference Numerals] (1) Neor detectionprimers (SNeoF and SNeoR), (2) KO verification 1 (KO Pro F SmaI and KOTerm R SmaI), (3) KO verification 2 (E2 KO ProF EcoRV and SNeoR), (4) KOverification 3 (SNeoF and E2 KO Term R EcoRV), (5) TaELO2 detection (E2HindIII and E2 XbaI); (B), the result of agarose electrophoresis in theevaluation of the transfectants by a PCR performed with template genomicDNA. [Brief Description of Reference Numerals] 1, 5, 9, 13, 17:transfectants; 2, 6, 10, 14, 18: wild-type strains; 3, 7, 11, 15, 19:samples using KONeor as a template; 4, 8, 12, 16: no template. Thenumbers (1) to (5) above the lane numbers represent the oligonucleotideprimer sets used.

FIG. 4 represents the result of confirming the copy numbers of TaELO2 bysouthern blotting in Example 2-9. [Brief Description of ReferenceNumerals] 1: genomic DNA (2.5 μg), BamHI treatment; 2: BglII treatment;3: EcoRI treatment; 4: EcoRV treatment; 5: HindIII treatment; 6: KpnItreatment; 7: SmaI treatment; 8: XbaI treatment; 9: positive control (aPCR product amplified with 1-ng E2 KO ProF EcoRV and E2 KO Term R EcoRV,containing TaELO2).

FIG. 5 represents the evaluation of TKONeor-introduced transfectants bysouthern blotting in Example 2-10. (A), a schematic view representingthe southern blotting performed for the detection of a wild-type alleleor a TKONeor-introduced mutant allele; (B), the result of southernblotting. [Brief Description of Reference Numerals] 1: T. aureumwild-type strain (2.5-μg genomic DNA); 2, 3: TKONeor-introducedtransfectants (2.5-μg genomic DNA); 4: positive control (a PCR productamplified with 50-ng E2 KO ProF EcoRV and E2 KO Term R EcoRV, containingTaELO2).

FIG. 6 represents the PCR evaluation performed in Example 2-12 by usingas a template the genomic DNA of the transfectant obtained byKOub600Hygr reintroduction. (A), the oligonucleotide primer set used.[Brief Description of Reference Numerals] (1) TaELO2 ORF detection(SNeoF and SNeoR), (2) KO verification (E2 KO Pro F EcoRV and ubi-hygroR); (B), the result of agarose electrophoresis in a PCR using theoligonucleotide primer set (1) for KO verification (arrows indicatetransfectants for which amplification of a specific product wasconfirmed, and that were assumed to be TaELO2-deficient homozygotes);(C) the result of agarose electrophoresis in a PCR performed for thetransfectants identified as TaELO2-deficient homozygotes using theoligonucleotide primer set (2) for TaELO2 ORF detection. [BriefDescription of Reference Numerals] 1: sample using KOub600Hygr as atemplate; 2: wild-type strain.

FIG. 7 represents the southern blotting evaluation of the transfectantsobtained by KOub600Hygr reintroduction in Example 2-12. (A), a schematicview representing the southern blotting performed for the detection of awild-type allele, a KONeor-introduced mutant allele, and aKOub600Hygr-introduced mutant allele; (B), the result of southernblotting. [Brief Description of Reference Numerals] 1, 9: wild-typestrains; 2-8 and 10-16: TaELO2-deficient homozygotes.

FIG. 8 represents the result of the southern blotting performed for thedetection of TaELO2 in Example 2-12. [Brief Description of ReferenceNumerals] 1: wild-type strain; 2-5: T TaELO2-deficient homozygotes.

FIG. 9 represents the result of the RT-PCR agarose gel electrophoresisperformed for the detection of TaELO2 mRNA in Example 2-12. [BriefDescription of Reference Numerals] 1-4: TaELO2-deficient homozygotes; 5:wild-type strain; 6-9: TaELO2-deficient homozygotes, using total RNA asa template (negative control); 10: wild-type strain, using total RNA asa template (negative control); 11: sample using wild-type strain genomicDNA as a template (positive control).

FIG. 10 represents the result of the comparison of the fatty acidcompositions of the wild-type strain and a TaELO2-deficient homozygotein Example 2-13.

FIG. 11 represents a plasmid containing the SV40 terminator sequencederived from a subcloned pcDNA 3.1 Myc-His vector.

FIG. 12 is a schematic view showing the primers used for fusion PCR, andthe product. The end product is the fused sequence of a Thraustochytriumaureum ATCC 34304-derived ubiquitin promoter and an artificialneomycin-resistant gene.

FIG. 13 represents a BglII cassette of the produced artificialneomycin-resistant gene.

FIG. 14 is a schematic view showing the primers used for fusion PCR, andthe product. The end product is the fused sequence of a Thraustochytriumaureum ATCC 34304-derived ubiquitin promoter and a pcDNA3.1/Hygro-derived hygromycin-resistant gene.

FIG. 15 represents a BglII cassette of the produced pcDNA3.1/Hygro-derived hygromycin-resistant gene.

FIG. 16 represents a plasmid containing a cloned Parietichytrium C20elongase sequence.

FIG. 17 represents a plasmid with a BglII site inserted into theParietichytrium C20 elongase sequence of the plasmid of FIG. 16.

FIG. 18 represents produced Parietichytrium C20 elongase gene targetingvectors (two vectors). The vectors have a neomycin-resistant gene(pRH85) or a hygromycin-resistant gene (pRH86) as a drug-resistancemarker.

FIG. 19 is a schematic view representing the positions of the PCRprimers used for the identification of the C20 elongase gene disruptedstrain of Parietichytrium sarkarianum SEK364, and the expected products.

FIG. 20 represents the C20 elongase gene disruption evaluation performedby a PCR using the Parietichytrium sarkarianum SEK364 genomic DNA as atemplate. [Description of Reference Numerals]+/+: Parietichytriumsarkarianum SEK364 wild-type strain; +/−: Parietichytrium sarkarianumSEK364-derived C20 elongase gene first allele homologous recombinant;−/−: Parietichytrium sarkarianum SEK364-derived C20 elongase genedisrupted strain.

FIG. 21 represents the result of the comparison of the fatty acidcompositions of the Parietichytrium sarkarianum SEK364 wild-type strainand the C20 elongase gene disrupted strain. Blank bar and solid barindicate the fatty acid compositions of the wild-type strain and thegene disrupted strain, respectively. All values are given as meanvalue±standard deviation.

FIG. 22 represents the proportions of the fatty acids of the C20elongase gene disrupted strain relative to the Parietichytriumsarkarianum SEK364 wild-type strain taken as 100%.

FIG. 23 is a schematic view of the primers used for fusion PCR, and theproduct. The end product is the fused sequence of Thraustochytriumaureum ATCC 34304-derived 18S rDNA, Thraustochytrium aureum ATCC34304-derived EF1α promoter, artificial neomycin-resistant gene, andThraustochytrium aureum ATCC 34304-derived EF1α terminator.

FIG. 24 represents a plasmid obtained by partial cloning of the DNAfragment joined in FIG. 23. The plasmid contains a partial sequence onthe 3′-end side of the EcoRI site of the Thraustochytrium aureum ATCC34304-derived 18S rDNA, the Thraustochytrium aureum ATCC 34304-derivedEF1α promoter, the artificial neomycin-resistant gene, and a partialsequence on the 5′-end side of the NcoI site of the Thraustochytriumaureum ATCC 34304-derived EF1α terminator.

FIG. 25 represents a produced Thraustochytrium aureum ATCC 34304 PKSpathway-associated gene orfA targeting vector. The vector has aneomycin-resistant gene as a drug-resistance marker.

FIG. 26 represents a plasmid containing the upstream sequence ofThraustochytrium aureum ATCC 34304 PKS pathway-associated gene orfA, aThraustochytrium aureum ATCC 34304-derived ubiquitin promoter, and ahygromycin-resistant gene.

FIG. 27 represents a produced Thraustochytrium aureum ATCC 34304 PKSpathway-associated gene orfA targeting vector. The vector has ahygromycin-resistant gene as a drug-resistance marker.

FIG. 28 is a schematic view representing the positions of the southernhybridization analysis probes used for the identification of the PKSpathway-associated gene orfA disrupted strain of Thraustochytrium aureumATCC 34304, and the expected gene fragment sizes.

FIG. 29 represents the evaluation of PKS pathway-associated gene orfAdisruption performed by southern hybridization using theThraustochytrium aureum ATCC 34304 genomic DNA. [Description ofReference Numerals] T. au: Thraustochytrium aureum ATCC 34304 wild-typestrain; +/−: Thraustochytrium aureum. ATCC 34304-derived PKSpathway-associated gene orfA first allele homologous recombinant; −/−:Thraustochytrium aureum ATCC 34304-derived PKS pathway-associated geneorfA disrupted strain.

FIG. 30 represents the result of the comparison of the fatty acidcompositions of the Thraustochytrium aureum ATCC 34304 wild-type strainand the PKS pathway-associated gene orfA disrupted strain. Blank bar andsolid bar indicate the fatty acid compositions of the wild-type strainand the gene disrupted strain, respectively. All values are given asmean value±standard deviation.

FIG. 31 represents the proportions of the fatty acids of the PKSpathway-associated gene orfA disrupted strain relative to theThraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

FIG. 32 is a schematic view representing the primers used for fusionPCR, and the product. The end product is the fused sequence ofThraustochytrium aureum ATCC 34304-derived ubiquitin promoter andpTracer-CMV/Bsd/lacZ-derived blasticidin-resistant gene.

FIG. 33 represents a pTracer-CMV/Bsd/lacZ-derived blasticidin-resistantgene BglII cassette.

FIG. 34 is a schematic view representing the primers used for fusionPCR, and the product. The end product is the fused sequence ofThraustochytrium aureum ATCC 34304-derived ubiquitin promoter andenhanced GFP gene (clontech).

FIG. 35 is a schematic view representing the primers used for fusionPCR, and the product. The end product is the fused sequence ofThraustochytrium aureum ATCC 34304-derived ubiquitin promoter, enhancedGFP gene (clontech), and pcDNA3.1 Zeo(+)-derived zeocin-resistant gene.

FIG. 36 represents a produced enhanced GFP-zeocin-resistant fused geneBglII cassette.

FIG. 37 represents a plasmid containing a cloned Thraustochytrium aureumATCC 34304 C20 elongase sequence and nearby sequences.

FIG. 38 represents a plasmid with the inserted BglII site after thecomplete deletion of the Thraustochytrium aureum ATCC 34304 C20 elongasesequence from the plasmid of FIG. 37.

FIG. 39 represents produced Thraustochytrium aureum ATCC 34304 C20elongase gene targeting vectors (two vectors). The vectors have ablasticidin-resistant gene (pRH43) or an enhanced GFP-zeocin-resistantfused gene (pRH54) as a drug-resistance marker.

FIG. 40 is a schematic view representing the positions of the southernhybridization analysis probes used for the identification of the C20elongase gene disrupted strain of the Thraustochytrium aureum ATCC 34304PKS pathway (orfA gene) disrupted strain, and the expected gene fragmentsizes.

FIG. 41 represents the evaluation of C20 elongase gene disruptionperformed by southern hybridization using the Thraustochytrium aureumATCC 34304 genomic DNA. [Description of Reference Numerals] T. au:Thraustochytrium aureum ATCC 34304 wild-type strain; −/−:Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene) andC20 elongase gene double disrupted strain.

FIG. 42 represents the result of the comparison of the fatty acidcompositions of the Thraustochytrium aureum ATCC 34304 wild-type strainand the PKS pathway (orfA gene) and C20 elongase gene double disruptedstrain. Blank bar and solid bar indicate the fatty acid compositions ofthe wild-type strain and the gene disrupted strain, respectively. Allvalues are given as mean value±standard deviation.

FIG. 43 represents the proportions of the fatty acids of the PKS pathway(orfA gene) and C20 elongase gene double disrupted strain relative tothe Thraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

FIG. 44 is a schematic view representing the primers used for fusionPCR, and the product. The end product is the fused sequence ofThraustochytrium aureum ATCC 34304-derived ubiquitin promoter,Saprolegnia diclina-derived? ω3 desaturase gene sequence, andThraustochytrium aureum ATCC 34304-derived ubiquitin terminator.

FIG. 45 represents the plasmid containing a KpnI site replacing one ofthe BglII sites in the blasticidin-resistant gene BglII cassette of FIG.33.

FIG. 46 represents a produced Saprolegnia diclina-derived ω3 desaturasegene expression plasmid. The plasmid has a blasticidin-resistant gene asa drug-resistance marker.

FIG. 47 is a schematic view representing the positions of the PCRprimers used for the confirmation of the genome insertion of theSaprolegnia diclina-derived ω3 desaturase gene.

FIG. 48 represents the evaluation of the transfectant strain derivedfrom the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene)disrupted strain. [Description of Reference Numerals] lanes 1 to 2:transfectants.

FIG. 49 represents the results of the comparison of the fatty acidcompositions of the control Thraustochytrium aureum ATCC 34304 PKSpathway (orfA gene) disrupted strain and the ω3 desaturase geneintroduced strain. Blank bar and solid bar indicate the fatty acidcompositions of the control strain and the ω3 desaturase gene introducedstrain, respectively. All values are given as mean value±standarddeviation.

FIG. 50 represents the proportions of the fatty acids of the ω3desaturase gene introduced strain relative to the Thraustochytriumaureum ATCC 34304 PKS pathway (orfA gene) disrupted strain taken as100%.

FIG. 51 is a diagram representing a pRH59 cloning the sequencecontaining the Thraustochytrium aureum ATCC 34304-derived C20 elongase.

FIG. 52 is a diagram representing a pRH64 cloning the sequencecontaining a BglII site in the Thraustochytrium aureum ATCC34304-derived C20 elongase.

FIG. 53 is a diagram representing a pRH65 containing aubiquitinpromoter-, neomycin-resistant gene-, and SV40 terminator-containingsequence cloned into the Thraustochytrium aureum ATCC 34304-derived C20elongase, and a pRH66 containing a ubiquitin promoter-,hygromycin-resistant gene-, and SV 40 terminator-containing sequencecloned into the Thraustochytrium aureum ATCC 34304-derived C20 elongase.

FIG. 54 represents the expected fragment sizes of the wild-type strainallele and knockout strains in a PCR.

FIG. 55 represents the detection results for the wild-type strain alleleand knockout strains in a PCR.

FIG. 56 represents the fatty acid compositions of the wild-type strainand the C20 elongase knockout strain. Blank bar and solid bar indicatethe fatty acid compositions of the wild-type strain and the strain,respectively.

FIG. 57 represents the result of the comparison of the fatty acidcompositions of the wild-type strain and the knockout strain.

FIG. 58 represents a plasmid containing a sequence from 1,071 bpupstream of the Δ4 desaturase gene to 1,500 bp within the Δ4 desaturasegene of the cloned Thraustochytrium aureum ATCC 34304 strain.

FIG. 59 represents a plasmid containing a BglII site inserted into thedeleted portion of the plasmid of FIG. 58 containing the 60 bp upstreamof the Δ4 desaturase gene and the 556-bp sequence containing the startcodon within the Δ4 desaturase gene (616 bp, SEQ ID NO: 205).

FIG. 60 represents produced Thraustochytrium aureum ATCC 34304 strain 44desaturase gene targeting vectors (two vectors). The vectors have ablasticidin resistant gene (pTM6) or an enhanced GFP-zeocin-resistantfused gene (pTM8) as a drug-resistance marker.

FIG. 61 is a schematic view representing the positions of the PCRprimers used for the identification of the Δ4 desaturase gene disruptedstrain of the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene)disrupted strain, and the expected product.

FIG. 62 represents the evaluation of 44 desaturase gene disruptionperformed by a PCR using the genomic DNA of the Thraustochytrium aureumATCC 34304 strain as a template. [Description of Reference Numerals]+/+:Thraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene)disrupted strain; +/−: 44 desaturase gene first allele homologousrecombinant derived from Thraustochytrium aureum ATCC 34304-derived PKSpathway (orfA gene) disrupted strain; −/−: Thraustochytrium aureum. ATCC34304-derived PKS pathway (orfA gene) and 44 desaturase gene doubledisrupted strain.

FIG. 63 represents the result of the comparison of the fatty acidcompositions of the Thraustochytrium aureum ATCC 34304 wild-type strain,and the PKS pathway (orfA gene) and 44 desaturase gene double disruptedstrain. Blank bar and solid bar indicate the fatty acid compositions ofthe wild-type strain and the gene disrupted strain, respectively.

FIG. 64 represents the proportions of the fatty acids of the PKS pathway(orfA gene) and 44 desaturase gene double disrupted strain relative tothe Thraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

FIG. 65 represents the evaluation of C20 elongase gene disruptionperformed by a PCR using the genomic DNA of the Parietichytrium sp.SEK358 strain as a template. [Description of Reference Numerals]+/+:Parietichytrium sp. SEK358 wild-type strain; −/−: Parietichytrium sp.SEK358 strain-derived C20 elongase gene disrupted strain.

FIG. 66 represents the result of the comparison of the fatty acidcompositions of the Parietichytrium sp. SEK358 wild-type strain, and theParietichytrium sp. SEK358 strain-derived C20 elongase gene disruptedstrain. Blank bar and solid bar indicate the fatty acid compositions ofthe wild-type strain and the gene disrupted strain, respectively.

FIG. 67 represents the proportions of the fatty acid compositions of theParietichytrium sp. SEK358 strain-derived C20 elongase gene disruptedstrain relative to the Parietichytrium sp. SEK358 wild-type strain takenas 100%. The diagonal line indicates that the fatty acid produced by theParietichytrium sp. SEK358 wild-type strain is below the detectionlimit.

FIG. 68 represents the evaluation of C20 elongase gene disruptionperformed by a PCR using the genomic DNA of the Parietichytrium sp.SEK571 strain as a template. [Description of Reference Numerals]+/+:Parietichytrium sp. SEK571 wild-type strain; −/−: Parietichytrium sp.SEK571 strain-derived C20 elongase gene disrupted strain.

FIG. 69 represents the result of the comparison of the fatty acidcompositions of the Parietichytrium sp. SEK571wild-type strain, and theParietichytrium sp. SEK571 strain-derived C20 elongase gene disruptedstrain. Blank bar and solid bar indicate the fatty acid compositions ofthe wild-type strain and the gene disrupted strain, respectively.

FIG. 70 represents the proportions of the fatty acids of theParietichytrium sp. SEK571 strain-derived C20 elongase gene disruptedstrain relative to the Parietichytrium sp. SEK571 wild-type strain takenas 100%.

FIG. 71 represents the multiple alignment of TΔ12d with the putativeamino acid sequences of the Δ12 desaturase genes derived fromThalassiosira pseudonana, Micromonas sp, and Phaeodactylum tricornutum.[Description of Reference Numerals] Underlined portion: histidine box.

FIG. 72 represents a GC analysis chart for the TΔ12d overexpressingstrain of the budding yeast Saccharomyces cerevisiae, and theproportions of fatty acid compositions.

FIG. 73 is a diagram representing a TΔ12d KO targeting vectorconstruction scheme.

FIG. 74 represents a scheme for the preparation of a homologousrecombination fragment for efficiently obtaining a homologousrecombinant by a split marker method.

FIG. 75 represents the result of the amplification of thehygromycin-resistant gene, blasticidin-resistant gene, and TΔ12d gene bya PCR performed by using the genomic DNAs of the wild-type strain, theTΔ12d first allele disrupted strain, and the TΔ12d disrupted strain (twoalleles are disrupted). [Description of Reference Numerals] M: λHindIIIdigest/φX174 HincII digest; W: wild-type; S1 to S3: 1st allele knock-outstrain; D1 to D3: 2nd allele knock-out strain.

FIG. 76 represents the result of the mRNA detection of thehygromycin-resistant gene, blasticidin-resistant gene, and TΔ12d gene bya RT-PCR for the wild-type strain, the TΔ12d first allele disruptedstrain, and the TΔ12d disrupted strain. [Description of ReferenceNumerals] M: λHindIII digest/φX174 HincII digest; W: wild-type; S1 toS3: 1st allele knock-out strain; D1 to D3: 2nd allele knock-out strain.

FIG. 77 represents the result of the southern blotting performed for thewild-type strain, the TΔ12d first allele disrupted strain, and the TΔ12ddisrupted strain.

FIG. 78 represents the result of the growth rate comparison by themeasurements of OD600 and dry cell weight for the wild-type strain, theTΔ12d first allele disrupted strain, and the TΔ12d disrupted strain.

FIG. 79 represents the proportions of the fatty acid compositions of thewild-type strain, the TΔ12d first allele disrupted strain, and the TΔ12ddisrupted strain. [Description of Reference Numerals] Asterisk:significant difference at p<0.01 (n=3).

FIG. 80 represents the fatty acid level per dry cell in the wild-typestrain, the TΔ12d first allele disrupted strain, and the TΔ12d disruptedstrain. [Description of Reference Numerals] Asterisk: significantdifference at p<0.01 (n=3).

FIG. 81 represents a plasmid containing a BamHI site inserted throughmodification of the Thraustochytrium aureum C20 elongase gene targetingvector (pRH43) of FIG. 39 with a blasticidin-resistant gene.

FIG. 82 represents a plasmid containing a KpnI site inserted throughmodification of the plasmid of FIG. 81.

FIG. 83 represents a produced Thraustochytrium aureum C20 elongase genetargeting and Saprolegnia diclina-derived ω3 desaturase expressionvector. The vector has a blasticidin-resistant gene as a drug-resistancemarker.

FIG. 84 is a schematic view representing the positions of the southernhybridization analysis probes used for the identification of the C20elongase gene disrupted and Saprolegnia diclina-derived ω3 desaturaseexpressing strain of the Thraustochytrium aureum PKS pathway (orfA gene)disrupted strain, and the expected gene fragment sizes.

FIG. 85 represents the evaluation of the C20 elongase gene disrupted andSaprolegnia diclina-derived ω3 desaturase expressing strain by southernhybridization using the Thraustochytrium aureum ATCC 34304 genomic DNA.[Description of Reference Numerals] PKSKO: Thraustochytrium aureum ATCC34304-derived PKS pathway (orfA gene) disrupted strain; +/−: C20elongase gene first allele homologous recombinant of theThraustochytrium aureum ATCC 34304-derived PKS pathway (orfA gene)disrupted strain; −/−: Thraustochytrium aureum-derived PKS pathway (orfAgene) and C20 elongase gene double disrupted and Saprolegniadiclina-derived ω3 desaturase expressing strain.

FIG. 86 represents the result of the comparison of the fatty acidcompositions of the Thraustochytrium aureum ATCC 34304 wild-type strain,and the PKS pathway (orfA gene) and C20 elongase gene double disruptedand Saprolegnia diclina-derived ω3 desaturase expressing strain. Blankbar and solid bar indicate the fatty acid compositions of the wild-typestrain and the gene disrupted strain, respectively.

FIG. 87 represents the proportions of the fatty acids of the PKS pathway(orfA gene) and C20 elongase gene double disrupted and Saprolegniadiclina-derived ω3 desaturase expressing strain relative to theThraustochytrium aureum ATCC 34304 wild-type strain taken as 100%.

FIG. 88 represents a base plasmid used for Saprolegnia diclina-derivedω3 desaturase expression vector production.

FIG. 89 represents a plasmid containing a Saprolegnia diclina-derived ω3desaturase expression KpnI cassette inserted into the plasmid of FIG.88.

FIG. 90 represents a Saprolegnia diclina-derived ω3 desaturaseexpression vector produced by inserting a hygromycin-resistant gene as adrug-resistance marker into the plasmid of FIG. 89.

FIG. 91 is a schematic view representing the positions of the PCRprimers used for the confirmation of the genome insertion of theSaprolegnia diclina-derived ω3 desaturase gene.

FIG. 92 represents the evaluation of the Parietichytrium sp. SEK571 C20elongase gene disrupted strain-derived transfectant strain. [Descriptionof Reference Numerals] Lanes 1 to 2: transfectants

FIG. 93 represents the result of the comparison of the fatty acidcompositions of the Parietichytrium sp. SEK571 wild-type strain, and theC20 elongase gene disrupted and Saprolegnia diclina-derived ω3desaturase expressing strain. Blank bar and solid bar indicate the fattyacid compositions of the wild-type strain and the transfectant strain,respectively.

FIG. 94 represents the proportions of the fatty acids of the C20elongase gene disrupted and Saprolegnia diclina-derived ω3 desaturaseexpressing strain relative to the Parietichytrium sp. SEK571wild-typestrain taken as 100%.

FIG. 95 is a diagram representing a pRH70 cloning a sequence containinga Schizochytrium-derived C20 elongase gene.

FIG. 96 is a diagram representing a pRH71 cloning a sequence containinga BglII site within the Schizochytrium-derived C20 elongase.

FIG. 97 is a diagram representing a pRH73 cloning a sequence containinga ubiquitin promoter, a neomycin-resistant gene, and an SV40 terminatorwithin the Schizochytrium-derived C20 elongase, and a pKS-SKO cloning asequence containing a ubiquitin promoter, a hygromycin-resistant gene,and an SV40 terminator within the Schizochytrium-derived C20 elongase.

FIG. 98 represents the expected fragment sizes of the wild-type strainallele and the knockout strains in a PCR.

FIG. 99 represents the PCR detection result for the wild-type strainallele and the knockout strain.

FIG. 100 represents the fatty acid compositions of the wild-type strainand the C20 elongase knockout strain. Blank bar and solid bar indicatethe fatty acid compositions of the wild-type strain and the strain,respectively.

FIG. 101 represents the result of the comparison of the fatty acidcompositions of the wild-type strain and the knockout strain.

MODE FOR CARRYING OUT THE INVENTION

The recent studies of the physiological activity and the pharmacologicaleffects of lipids have elucidated the conversion of unsaturated fattyacids into various chemical substances, and the roles of unsaturatedfatty acids in the unsaturated fatty acid metabolism. Particularlyconsidered important in relation to disease is the nutritionallypreferred proportions of saturated fatty acids, monounsaturated fattyacids, and unsaturated fatty acids, and the proportions of fishoil-derived ω3 series (also known as the n-3 series) fatty acids such aseicosapentaenoic acid and docosahexaenoic acid, and plant-derived ω6series (also known as the n-6 series) fatty acids as represented bylinoleic acid. Because animals are deficient in fatty acid desaturases(desaturases) or have low levels of fatty acid desaturases, someunsaturated fatty acids need to be ingested with food. Such fatty acidsare called essential fatty acids (or vitamin F), which include linoleicacid (LA), γ-linolenic acid (GLA), and arachidonic acid (AA or ARA).

Unsaturated fatty acid production involves enzymes called fatty aciddesaturases (desaturases). The fatty acid desaturases (desaturases) areclassified into two types: (1) those creating a double bond (also calledan unsaturated bond) at a fixed position from the carbonyl group of afatty acid (for example, 49 desaturase creates a double bond at the 9thposition as counted from the carbonyl side), and (2) those creating adouble bond at a specific position from the methyl end of a fatty acid(for example, ω3 desaturase creates a double bond at the 3rd position ascounted from the methyl end). It is known that the biosynthesis ofunsaturated fatty acid involves the creation of a double bond by thedesaturase (unsaturation), and the repeated elongation of the chainlength by several different elongases. For example, Δ9 desaturasesynthesizes oleic acid (OA) by unsaturating the stearic acid eithersynthesized in the body from palmitic acid or ingested directly from theoutside of the body. Δ6, Δ5, and Δ4 desaturases are fatty aciddesaturases (desaturases) essential for the syntheses of polyunsaturatedfatty acids such as arachidonic acid (AA), eicosapentaenoic acid (EPA),and docosahexaenoic acid (DHA).

The Labyrinthulomycetes, a member of stramenopile, has two families:Thraustochytrium (Thraustochytriaceae) and Labyrinthulaceae. Thesemicroorganisms are known to accumulate polyunsaturated fatty acids suchas arachidonic acid, EPA, DTA, DPA, and DHA.

The present invention is concerned with a stramenopile transformationmethod whereby stramenopile genes are disrupted and/or expressionthereof is inhibited by genetic engineering. Specifically, the presentinvention developed and provides a transformation method for disruptinggenes associated with fatty acid biosynthesis and/or inhibitingexpression thereof, a method for modifying the fatty acid composition ofa stramenopile with the use of the transformation method, a method forhighly accumulating fatty acids in a stramenopile, a stramenopile havingan enhanced unsaturated fatty acid content, and a method for producingunsaturated fatty acid from the unsaturated fatty acid content-enhancedstramenopile.

The present invention includes manipulating the enzymes of thestramenopile elongase/desaturase pathway to change the fatty acidcomposition produced by a stramenopile. Specifically, the presentinvention enables modification of the fatty acid composition produced bystramenopile through (1) disruption of a fatty acid chain elongase geneand/or inhibition of expression thereof, (2) disruption of a polyketidesynthase gene and/or inhibition of expression thereof, (3) disruption ofa fatty acid desaturase and/or inhibition of expression thereof, (3)disruption of two of or all of a polyketide synthase gene, a fatty acidchain elongase gene, and a fatty acid desaturase and/or inhibition ofexpression thereof, (4) disruption of a fatty acid chain elongase geneand/or inhibition of expression thereof, and introduction of a fattyacid desaturase gene, (5) disruption of a polyketide synthase geneand/or inhibition of expression thereof, and introduction of a fattyacid desaturase gene, (6) disruption of a fatty acid desaturase and/orinhibition of expression thereof, and introduction of a fatty aciddesaturase gene, (6) disruption of two of or all of a polyketidesynthase gene, a fatty acid chain elongase gene, and a fatty aciddesaturase and/or inhibition of expression thereof, and introduction ofa fatty acid desaturase gene.

The present invention is described below in more detail.

[Microorganism]

The microorganisms used in the fatty acid modification method of thepresent invention are not particularly limited, as long as themicroorganisms are stramenopiles considered to undergo modification ofthe fatty acid composition through disruption of genes associated withfatty acid biosynthesis and/or inhibition of expression thereof.Particularly preferred microorganisms are those belonging to the classLabyrinthulomycetes. Examples of the Labyrinthulomycetes include thoseof the genus Labyrinthula, Althornia, Aplanochytrium, Japonochytrium,Labyrinthuloides, Schizochytrium, Thraustochytrium, Ulkenia,Aurantiochytrium, Oblongichytrium, Botryochytrium, Parietichytrium, andSicyoidochytrium.

Of note, Labyrinthuloides and Aplanochytrium are regarded as beingsynonymous among some scholars (Leander, Celeste A. & David Porter,Mycotaxon, vol. 76, 439-444 (2000)).

The Labyrinthulomycetes used in the present invention are preferablymicroorganisms belonging to the genus Thraustochytrium and the genusParietichytrium, particularly preferably Thraustochytrium aureum,Parietichytrium sarkarianum, and Thraustochytrium roseum. Specificexamples include strains of Thraustochytrium aureum ATCC 34304,Parietichytrium sarkarianum SEK 364 (FERM BP-11298), Thraustochytriumroseum ATCC 28210, Parietichytrium sp. SEK358 (FERM BP-11405), andParietichytrium sp. SEK571 (FERM BP-11406). Thraustochytrium aureum ATCC34304 and Thraustochytrium roseum ATCC 28210 are deposited at the ATCC,and are commonly available. The Parietichytrium sarkarianum SEK364strain was obtained from the surface water collected at the mouth offukidougawa on Ishigakijima. The water (10 ml) was placed in a testtube, and left unattended at room temperature after adding pine pollens.After 7 days, the pine pollens were applied to a sterile agar medium (2g glucose, 1 g peptone, 0.5 g yeast extract, 0.2 g chloramphenicol, 15 gagar, distilled water 100 mL, sea water 900 mL). Colonies appearingafter 5 days were isolated and cultured. This was repeated several timesto isolate the cells. This strain has been internationally deposited,and is available from The National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary(Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki)(accession number: FERM BP-11298; Sep. 24, 2010). The Parietichytriumsp. SEK358 strain was isolated from the cells cultured as above from thesea water sample collected at the mouth of Miyaragawa on Ishigakijima.This strain has been internationally deposited, and is available fromThe National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary (Tsukuba Center, Chuou Dairoku,1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number: FERM BP-11405;Aug. 11, 2011). The Parietichytrium sp. SEK571 strain was isolated fromthe cells cultured as above from the sea water sample collected at themouth of Shiiragawa on Iriomotejima. This strain has beeninternationally deposited, and is available from The National Instituteof Advanced Industrial Science and Technology, International PatentOrganism Depositary (Tsukuba Center, Chuou Dairoku, 1-1-1, Higashi,Tsukuba-shi, Ibaraki) (accession number: FERM BP-11406; Aug. 11, 2011).The Schizochytrium sp. TY12Ab strain was isolated from the cellscultured as above from the dead leaves collected on the coast ofTanegashima. This strain has been internationally deposited, and isavailable from The National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary (Tsukuba Center,Chuou Dairoku, 1-1-1, Higashi, Tsukuba-shi, Ibaraki) (accession number:FERM ABP-11421; Sep. 29, 2011). Then, the RECEIPT IN THE CASE OF ANORIGINAL DEPOSIT (FERM BP-11421) was issued by International PatentOrganism Depositary on Nov. 30, 2011

[Genes Associated with Fatty Acid Biosynthesis]

In the present invention, the genes associated with fatty acidbiosynthesis are not particularly limited, as long as the genes aregenes of enzymes associated with the fatty acid biosynthesis instramenopile, particularly the Labyrinthulomycetes. Examples of suchgenes include polyketide synthase gene, fatty acid chain elongase gene,and fatty acid desaturase gene. In the present invention, one of or bothof these genes are subject to the disruption or inhibition of expressionby genetic engineering. Here, the target of the gene disruption and/orinhibition of expression is, for example, the open reading frame, when,for example, the fatty acid produced by the polyketide synthase in astramenopile is not the desired fatty acid. In the case of the fattyacid chain elongase, the target is the gene associated with an enzymethat converts the desired fatty acid into an other fatty acid. Forexample, when eicosapentaenoic acid (EPA) is the desired product, thegene of the fatty acid chain elongase associated with the conversion ofeicosapentaenoic acid into docosapentaenoic acid (DPA), specifically C20elongase gene may be disrupted and/or expression thereof may beinhibited. In the case of the fatty acid desaturase, the target is thegene associated with the enzyme that converts the desired fatty acidinto an other fatty acid. For example, when oleic acid is the desiredproduct, the gene of the fatty acid desaturase associated with theconversion of oleic acid into linoleic acid, specifically 412 desaturasegene may be disrupted and/or expression thereof may be inhibited.Further, two of or all of the polyketide synthase gene, the fatty acidchain elongase gene, and the fatty acid desaturase may be disruptedand/or expression thereof may be inhibited according to the desiredfatty acid.

Further, a gene associated with fatty acid biosynthesis may beintroduced into a transfectant strain produced by disrupting a geneand/or inhibiting expression thereof by genetic engineering as above.Here, the introduced gene is a gene associated with the enzyme thatperforms biosynthesis of the desired fatty acid. For example, wheneicosapentaenoic acid is the desired product, a gene of the fatty aciddesaturase that converts arachidonic acid (AA) into eicosapentaenoicacid, specifically ω3 desaturase gene may be introduced.

[Polyketide Synthase and Fatty Acid Chain Elongase]

Polyketide synthase (PKS) is an enzyme that catalyzes the multiplecondensation reactions of a starter substrate (acetyl-CoA, fatty acidCoA ester, benzoyl CoA, coumaroyl CoA) with an extender substrate (suchas malonyl CoA), and the enzyme is generally known to be involved in thebiosyntheses of secondary metabolites in organisms such as plants andfungi. Involvement in the biosynthesis of polyunsaturated fatty acid isalso reported in some species of organisms. For example, the marinebacteria Shewanella produce eicosapentaenoic acid (EPA) with this enzyme(Non-Patent Document 8). In some species of stramenopile, the polyketidesynthase is known to be involved in the biosynthesis of polyunsaturatedfatty acid, and the gene sequence has been elucidated in theLabyrinthulomycetes. For example, as described in Patent Document 7, thepolyketide synthase gene of the genus Schizochytrium of Labyrinthula hasthree open reading frames, OrfA, OrfB, and OrfC. Further, as describedin Patent Document 8, the polyketide synthase gene of the genus Ulkeniaof Labyrinthula is considered to have three open reading frames.

The fatty acid chain elongase of the present invention is notparticularly limited, as long as it extends the chain length of a fattyacid. Preferred examples include C18 elongase gene, and C20 elongasegene. The C18 elongase gene and the C20 elongase gene extend fatty acidsof 18 and 20 carbon atoms, respectively, in two-carbon units to producefatty acids of 20 and 22 carbon atoms. These fatty acid chain elongasesare found in a wide range of organisms, including stramenopiles, and in,for example, the genus Thraustochytrium of Labyrinthulomycetes, asreported in Non-Patent Document 9. The C18 elongase catalyzes theconversion of γ-linolenic acid (GLA) to dihomo-γ-linolenic acid (DGLA),and the conversion of stearidonic acid (STA) into eicosatetraenoic acid(ETA). The C20 elongase catalyzes the conversion of arachidonic acid(AA) into docosatetraenoic acid (DTA), and the conversion ofeicosapentaenoic acid (EPA) into n-3 docosapentaenoic acid (DPA,22:5n-3).

It follows from this that when the desired product is, for example,stearidonic acid (STA), a gene of the fatty acid chain elongaseassociated with the conversion of stearidonic acid into eicosatetraenoicacid (ETA), specifically C18 elongase gene may be disrupted and/orexpression thereof may be inhibited. When the desired product is, forexample, eicosapentaenoic acid (EPA), a gene of the fatty acid chainelongase associated with the conversion of the eicosapentaenoic acidinto docosapentaenoic acid (DPA), specifically C20 elongase gene may bedisrupted and/or expression thereof may be inhibited. Further, when thefatty acid biosynthesized with the polyketide synthase in a stramenopileis not the desired fatty acid, the polyketide synthase gene may bedisrupted and/or expression thereof may be inhibited. As reported inNon-Patent Document 5, a strain of the genus Schizochytrium ofLabyrinthula loses the ability to biosynthesize docosahexaenoic acidafter the disruption of the polyketide synthase gene, and cannot grow inmedia unless supplemented with polyunsaturated fatty acid. In thepresent invention, however, some species of Labyrinthula, even with thedisrupted polyketide synthase gene, are able to grow in media withoutadding polyunsaturated fatty acid, and the desired polyunsaturated fattyacid can thus be obtained by disrupting the gene or inhibiting geneexpression in the manner described above.

[Fatty Acid Desaturase]

The fatty acid desaturase (desaturase) of the present invention is notparticularly limited, as long as it functions as a fatty aciddesaturase. The origin of the fatty acid desaturase gene is notparticularly limited, and may be, for example, animals and plants.Examples of the preferred fatty acid desaturase genes include Δ4desaturase gene, Δ5 desaturase gene, Δ6 desaturase gene, Δ12 desaturasegene, and ω3 desaturase gene, and these may be used either alone or incombination. The Δ4 desaturase gene, Δ5 desaturase gene, Δ6 desaturasegene, and Δ12 desaturase gene form an unsaturated bond at carbon 4, 5,6, and 12, respectively, as counted from the carbon atom of the terminalcarboxyl group (delta end) of the fatty acid. A specific example ofthese fatty acid desaturase genes is the microalgae-derived Δ4desaturase gene (Non-Patent Document 6). Specific examples of Δ5desaturase include T. aureum-derived Δ5 desaturase, and Δ5 desaturasesderived from Thraustochytrium sp. ATCC 26185, Dictyostelium discoideum,Rattus norvegicus, Mus musculus, Homo sapiens, Caenorhabditis elegans,and Leishmania major. Examples of 412 desaturase include Pinguiochrysispyriformis-derived Δ12 desaturase, and fungus- and protozoa-derived Δ12desaturases. The ω3 desaturase forms a double bond at the third positionas counted from the methyl terminal of the fatty acid carbon chain.Examples include Saprolegnia-derived ω3 desaturase (Non-Patent Document10). The Δ5 desaturase catalyzes, for example, the conversion ofdihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA), and theconversion of eicosatetraenoic acid (ETA) to eicosapentaenoic acid(EPA). Δ6 desaturase catalyzes, for example, the conversion of linoleicacid (LA) to γ-linolenic acid (GLA), and the conversion of α-linolenicacid (ALA) to stearidonic acid (STA). The ω3 desaturase catalyzes theconversion of arachidonic acid to eicosapentaenoic acid. Linoleic acid(LA) is produced from oleic acid (OA) by the action of Δ12 desaturase.

[Product Unsaturated Fatty Acid]

The unsaturated fatty acid produced by the fatty acid desaturaseexpressed in a stramenopile is, for example, an unsaturated fatty acidof 18 to 22 carbon atoms. Preferred examples include docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA), though the preferredunsaturated fatty acids vary depending on the types of the fatty aciddesaturase and the fatty acid substrate used. Other examples includeα-linolenic acid (ALA), octadecatetraenoic acid (OTA, 18:4n-3),eicosatetraenoic acid (ETA, 20:4n-3), n-3 docosapentaenoic acid (DPA,22:5n-3), tetracosapentaenoic acid (TPA, 24:5n-3), tetracosahexaenoicacid (THA, 24:6n-3), linoleic acid (LA), γ-linolenic acid (GLA),eicosatrienoic acid (20:3n-6), arachidonic acid (AA), and n-6docosapentaenoic acid (DPA, 22:5n-6).

[Gene Source of Enzyme Associated with Fatty Acid Biosynthesis]

The organisms that can be used as the gene sources of the polyketidesynthase, fatty acid chain elongase, and/or fatty acid desaturase in thepresent invention are not limited to particular genuses, species, orstrains, and may be any organisms having an ability to producepolyunsaturated fatty acids. For example, in the case of microorganisms,such organisms are readily available from microorganism depositaryauthorities. Examples of such microorganisms include the bacteriaMoritella marina MP-1 strain (ATCC15381) of the genus Moritella. Thefollowing describes a method using this strain as an example ofdesaturase and elongase gene sources. The method, however, is alsoapplicable to the isolation of the constituent desaturase and elongasegenes from all biological species having the desaturase/elongasepathway.

Isolation of the desaturase and/or elongase gene from the MP-1 strainrequires estimation of a conserved region in the amino acid sequence ofthe target enzyme gene. For example, in desaturase, it is known that asingle cytochrome b5 domain and three histidine boxes are conservedacross biological species, and that elongase has two conserved histidineboxes across biological species. More specifically, the conserved regionof the target enzyme can be estimated by the multiple alignmentcomparison of the known amino acid sequences of the desaturase orelongase genes derived from various biological species using the clustalw program (Non-Patent Document 7). It is also possible to estimateconserved regions specific to desaturase and/or elongase having the samesubstrate specificity by the multiple alignment comparison of the aminoacid sequences of desaturase or elongase genes having the same substratespecificity in the desaturase and/or elongase derived from known otherorganisms. Various degenerate oligonucleotide primers are then producedbased on the estimated conserved regions, and the partial sequence ofthe target gene derived from the MP-1 strain is amplified using an MP-1strain-derived cDNA library as a template, by using methods such as PCRand RACE. The resulting amplification product is cloned into a plasmidvector, and the base sequence is determined using an ordinary method.The sequence is then compared with a known enzyme gene to confirmisolation of a part of the target enzyme gene from the MP-1 strain. Thefull-length target enzyme gene can be obtained by hybridizationscreening using the obtained partial sequence as a probe, or by the RACEtechnique using the oligonucleotide primers produced from the partialsequence of the target gene.

The polyketide synthase can be cloned by using an ordinary method, usingthe PUFA PKS sequence of Patent Document 7 as a reference.

[Other Gene Sources]

Reference should be made to Non-Patent Document 11 or 12 for GFP (GreenFluorescent Protein), Patent Document 6 for EGFP (enhanced GFP), andNon-Patent Document 13 for neomycin-resistant gene.

[Disruption of Gene Associated with Fatty Acid Biosynthesis inStramenopile]

The stramenopile gene associated with fatty acid biosynthesis may bedisrupted by using conventional gene disruption methods used formicroorganisms. An example of such a method is the transformationintroducing a recombinant expression vector into a cell.

For example, for the disruption of a Thraustochytrium aureum gene,genomic DNA is extracted from a Thraustochytrium aureum by using anordinary method, and a genome library is created. Then, genome walkingprimers are set using the DNA sequence of the target gene to bedisrupted, and a PCR is run using the produced genome library as atemplate to obtain the upstream and downstream sequences of the targetgene of Thraustochytrium aureum. These sequences are flanked on bothsides to provide homologous recombination regions for gene disruption,and a drug marker gene is inserted therebetween for selection. The DNAis then linearized, and introduced into a Thraustochytrium aureum usinga gene-gun technique, and the cells are cultured for about 1 week on adrug-containing plate. By using an ordinary method, genomic DNA isextracted from cells that have acquired drug resistance, and strainsthat underwent homologous recombination are identified by PCR orsouthern hybridization. Because the Thraustochytrium aureum is adiploid, the procedures from the introduction of the linearized DNA intothe cells using a gene-gun technique to the identification of homologousrecombinant strains are repeated twice. In this way, a Thraustochytriumaureum with the disrupted target gene can be obtained. When two or moretarget genes are present, a strain with the disrupted multiple targetgenes can be obtained by repeating the foregoing procedures. Here,because the Thraustochytrium aureum is a diploid, two selection markersneed to be prepared for each gene.

For the disruption of, for example, the C20 elongase of Parietichytriumsarkarianum, genomic DNA is extracted from a Parietichytrium species byusing an ordinary method, and the genome is decoded. Then, a search ismade for a gene sequence highly homologous to a known C20 elongase gene,and the gene sequence is amplified by PCR from the start codon to thestop codon. This is followed by insertion of a restriction enzyme siteat substantially the center of the gene sequence by using a mutagenesismethod, and insertion of a drug marker gene cassette to the restrictionenzyme site for selection. The DNA is linearized, and introduced into aParietichytrium sarkarianum SEK364 using a gene-gun technique. The cellsare then cultured for about 1 week on a drug-containing plate. By usingan ordinary method, genomic DNA is extracted from cells that haveacquired drug resistance, and strains that underwent homologousrecombination are identified by PCR. Because the Parietichytriumsarkarianum SEK364 is a diploid, the procedures from the introduction ofthe linearized DNA into the cells using a gene-gun technique to theidentification of the homologous recombinant strains are repeated twice.In this way, a Parietichytrium sarkarianum SEK364 with the disrupted C20elongase gene can be obtained. Here, because the Parietichytriumsarkarianum SEK364 is a diploid, two selection markers need to beprepared. For the disruption of, for example, the Δ4 desaturase of aThraustochytrium aureum ATCC 34304-derived OrfA disrupted strain,genomic DNA is extracted from a Thraustochytrium aureum ATCC 34304 byusing an ordinary method, and the genome is decoded. Then, a search ismade for a gene sequence highly homologous to a known 44 desaturase, andthe gene sequence is amplified by PCR from the upstream region to aregion in the vicinity of the stop codon. By using a mutagenesis method,a restriction enzyme site is inserted at the same time as deleting apart of the ORF containing the start codon, and a drug marker genecassette is inserted to the restriction enzyme site for selection. TheDNA is linearized, and introduced into a Thraustochytrium aureum ATCC34304-derived OrfA disrupted strain by using a gene-gun technique. Thecells are then cultured for about 1 week on a drug-containing plate. Byusing an ordinary method, genomic DNA is extracted from cells that haveacquired drug resistance, and strains that underwent homologousrecombination are identified by PCR. Because the Thraustochytrium aureumATCC 34304 is a diploid, the procedures from the introduction of thelinearized DNA using a gene-gun technique to the identification ofhomologous recombinant strains are repeated twice. In this way, aThraustochytrium aureum ATCC 34304-derived OrfA disrupted strain withthe disrupted Δ4 desaturase gene can be obtained. Here, because theThraustochytrium aureum ATCC 34304 is a diploid, two selection markersneed to be prepared.

The C20 elongase gene sequence of Thraustochytrium aureum was used fordisrupting the C20 elongase of Thraustochytrium roseum. Genomic DNA isextracted from a Thraustochytrium aureum by using an ordinary method,and the C20 elongase gene is amplified from the start codon to the stopcodon by PCR. A restriction enzyme site is inserted to substantially thecenter of the gene sequence by using a mutagenesis method, and a drugmarker gene cassette is inserted to the restriction enzyme site forselection. The DNA is linearized, and introduced into a Thraustochytriumroseum by using a gene-gun technique. The cells are then cultured forabout 1 week on a drug-containing plate. By using an ordinary method,genomic DNA is extracted from cells that have acquired drug resistance,and strains that underwent homologous recombination are identified byPCR. Because the Thraustochytrium roseum is a diploid, the proceduresfrom the introduction of the linearized DNA using a gene-gun techniqueto the identification of the homologous recombinant strain are repeatedtwice. In this way, a Thraustochytrium roseum with the disrupted C20elongase gene can be obtained. Here, because the Thraustochytrium roseumis a diploid, two selection markers need to be prepared.

Details of the disruption of stramenopile genes associated with fattyacid biosynthesis according to the present invention will bespecifically described later in Examples. The stramenopile subject totransformation is not particularly limited, and those belonging to theclass Labyrinthulomycetes can preferably be used, as described above.

For example, for the disruption of the C20 elongase of theParietichytrium sp. SEK358 strain, the Parietichytrium C20 elongase genetargeting vector produced in Example 3-6 was used. The DNA islinearized, and introduced into a Parietichytrium sp. SEK358 strain byusing a gene-gun technique. The cells are then cultured for about 1 weekon a drug-containing plate. By using an ordinary method, genomic DNA isextracted from cells that have acquired drug resistance, and strainsthat underwent homologous recombination were identified by PCR (seeExample 9). For the disruption of, for example, the C20 elongase of theParietichytrium sp. SEK571 strain, the Parietichytrium C20 elongase genetargeting vector produced in Example 3-6 was used. The DNA islinearized, and introduced into a Parietichytrium sp. SEK571 strain byusing a gene-gun technique. The cells are then cultured for about 1 weekon a drug-containing plate. By using an ordinary method, genomic DNA wasextracted from cells that had acquired drug resistance, and thehomologous recombinant strain was identified by PCR (see Example 10).

The expression vector is not particularly limited, and a recombinantexpression vector with an inserted gene may be used. The vehicle used toproduce the recombinant expression vector is not particularly limited,and, for example, a plasmid, a phage, and a cosmid may be used. A knownmethod may be used for the production of the recombinant expressionvector. The vector is not limited to specific types, and may beappropriately selected from vectors expressible in a host cell.Specifically, the expression vector may be one that is produced byincorporating the gene of the present invention into a plasmid or othervehicles with a promoter sequence appropriately selected according tothe type of the host cell for reliable expression of the gene. Thevector may be a cyclic or a linear vector. The expression vectorpreferably includes at least one selection marker. Examples of suchselection markers include auxotrophic markers, drug-resistance markers,fluorescent protein markers, and fused markers of these. Examples of theauxotrophic markers include dihydrofolate reductase genes. Examples ofthe drug-resistance markers include neomycin-resistant genes,hygromycin-resistant genes, blasticidin-resistant genes, andzeocin-resistant genes. Examples of the fluorescent protein markersinclude GFPs, and enhanced GFPs (EGFPs). Examples of the fused markersinclude fused markers of fluorescent protein markers and drug-resistancemarkers, specifically, for example, GFP-fused zeocin-resistant genes.These selection markers allow for confirmation of whether thepolynucleotide according to the present invention has been introducedinto a host cell, or whether the polynucleotide is reliably expressed inthe host cell. Alternatively, the fatty acid desaturase according to thepresent invention may be expressed as a fused polypeptide. For example,the fatty acid desaturase according to the present invention may beexpressed as a GFP-fused polypeptide, using GFP as a marker.

Preferably, electroporation or a gene gun is used as the method of geneintroduction for the gene disruption. In the present invention, thedisruption of the gene associated with fatty acid biosynthesis changesthe fatty acid composition of the cell from that before the genedisruption. Specifically, the fatty acid composition is modified by thedisruption of the gene associated with fatty acid biosynthesis. Astramenopile with the disrupted fatty acid biosynthesis-related enzymegene can produce the desired fatty acid in greater amounts when furtherintroduced with a fatty acid desaturase gene. Preferably, an ω3desaturase gene is introduced as the fatty acid desaturase gene.

The stramenopile transformation produces a stramenopile (microorganism)in which the composition of the fatty acid it produces is modified. Thestramenopile with the disrupted gene associated with fatty acidbiosynthesis can be used for, for example, the production of unsaturatedfatty acids. Unsaturated fatty acid production is possible with thestramenopile that has been modified to change its produced fatty acidcomposition as above, and other conditions, including steps, equipment,and instruments are not particularly limited. The unsaturated fatty acidproduction includes the step of culturing a microorganism that has beenmodified to change its produced fatty acid composition by the foregoingmodification method, and the microorganism is used with its medium toproduce unsaturated fatty acids.

The cell culture conditions (including medium, culture temperature, andaeration conditions) may be appropriately set according to such factorsas the type of the cell, and the type and amount of the unsaturatedfatty acid to be produced. As used herein, the term “unsaturated fattyacids” encompasses substances containing unsaturated fatty acids, andattributes such as the content, purity, shape, and composition are notparticularly limited. Specifically, in the present invention, the cellor its medium itself having a modified fatty acid composition may beregarded as unsaturated fatty acids. Further, a step of purifying theunsaturated fatty acids from such cells or media also may be included. Aknown method of purifying unsaturated fatty acids and other lipids(including conjugate lipids) may be used for the purification of theunsaturated fatty acids.

[Method of Highly Accumulating Unsaturated Fatty Acid in Stramenopile]

Accumulation of unsaturated fatty acids in stramenopile is realized byculturing the transformed stramenopile of the present invention. Forexample, the culture is performed using a common solid or liquid medium.The type of medium used is not particularly limited, as long as it isone commonly used for culturing Labyrinthulomycetes, and that contains,for example, a carbon source (such as glucose, fructose, saccharose,starch, and glycerine), a nitrogen source (such as a yeast extract, acorn steep liquor, polypeptone, sodium glutamate, urea, ammoniumacetate, ammonium sulfate, ammonium nitrate, ammonium chloride, andsodium nitrate), and an inorganic salt (such as potassium phosphate)appropriately combined with other necessary components. Particularlypreferably, a yeast extract/glucose medium (GY medium) is used. Theprepared medium is adjusted to a pH of 3.0 to 8.0, and used after beingsterilized with an autoclave or the like. The culture may be performedby aerated stirred culture, shake culture, or static culture at 10 to40° C., preferably 15 to 35° C., for 1 to 14 days.

For the collection of the produced unsaturated fatty acids, thestramenopile is grown in a medium, and the intracellular lipids (oil andfat contents with the polyunsaturated fatty acids, or thepolyunsaturated fatty acids) are released by processing themicroorganism cells obtained from the medium. The lipids are thencollected from the medium containing the released intracellular lipids.Specifically, the cultured stramenopile is collected by using a methodsuch as centrifugation. The cells are then disrupted, and theintracellular fatty acids are extracted using a suitable organic solventaccording to an ordinary method. Oil and fat with the enhancedpolyunsaturated fatty acid content can be obtained in this manner.

In the present invention, the composition of the fatty acids produced bya stramenopile is modified by culturing a stramenopile transformedthrough disruption of genes associated with fatty acid biosynthesis,and/or inhibition of expression thereof, specifically disruption of thepolyketide synthase, the fatty acid chain elongase, and/or the fattyacid desaturase gene, and/or inhibition of expression of these genes.Because the genes associated with fatty acid biosynthesis are disruptedand/or expression thereof is inhibited, the desired fatty acid can beaccumulated in the stramenopile without being converted into other fattyacids. Further, by introducing the gene associated with fatty aciddesaturase into a stramenopile transformed through gene disruptionand/or inhibition of gene expression, the ability to convert theprecursor fatty acid of the desired fatty acid into the desired fattyacid can be enhanced, and the desired fatty acid is accumulated.

The unsaturated fatty acids of the present invention encompass variousdrugs, foods, and industrial products, and the applicable areas of theunsaturated fatty acids are not particularly limited. Examples of thefood containing oil and fat that contain the unsaturated fatty acids ofthe present invention include foods with health claims such assupplements, and food additives. Examples of the industrial productsinclude feeds for non-human organisms, films, biodegradable plastics,functional fibers, lubricants, and detergents.

The present invention is described below in more detail based onexamples. Note, however, that the present invention is in no way limitedby the following examples.

Example 1

[Labyrinthulomycetes, Culture Method, and Preservation Method]

(1) Strains Used in the Present Invention

Thraustochytrium aureum ATCC 34304 and Thraustochytrium roseum ATCC28210 were obtained from ATCC. Parietichytrium sarkarianum SEK364 (FERNBP-11298), Parietichytrium sp. SEK358 (FERM BP-11405), andParietichytrium sp. SEK571 (FERN BP-11406) were obtained from KonanUniversity, Faculty of Science and Engineering. Schizochytrium sp.TY12Ab (FERN BP-11421) was obtained from University of Miyazaki, Facultyof Agriculture.

(2) Medium Composition

i. Agar Plate Medium Composition

PDA Agar Plate Medium

A 0.78% (w/v) potato dextrose agar medium (Nissui Pharmaceutical Co.,Ltd.), 1.75% (w/v) Sea Life (Marine Tech), and 1.21% (w/v) agar powder(nacalai tesque) were mixed, and sterilized with an autoclave at 121° C.for 20 min. After sufficient cooling, ampicillin sodium (nacalai tesque)was added in a final concentration of 100 μg/ml to prevent bacterialcontamination. The medium was dispensed onto a petri dish, and allowedto stand on a flat surface to solidify.

ii. Liquid Medium Composition

GY Liquid Medium

3.18% (w/v) glucose (nacalai tesque), 1.06% (w/v) dry yeast extract(nacalai tesque), and 1.75% (w/v) Sea Life (Marine Tech) were mixed, andsterilized with an autoclave at 121° C. for 20 min. Then, 100 μg/mlampicillin sodium (nacalai tesque) was added.

PD Liquid Medium

0.48% (w/v) potato dextrose (Difco), and 1.75% (w/v) Sea Life (MarineTech) were mixed, and sterilized with an autoclave at 121° C. for 20min. Then, 100 μg/ml ampicillin sodium (nacalai tesque) was added.

(3) Culture Method

i. Agar Plate Culture

Labyrinthula cells were inoculated using a platinum loop or a spreader,and static culture was performed at 25° C. to produce colonies.Subcultures were produced by collecting the colonies with a platinumloop, suspending the collected colonies in a sterilized physiologicalsaline, and applying the suspension using a platinum loop or a spreader.As required, the cells on the plate were inoculated in a liquid mediumfor conversion into a liquid culture.

ii. Liquid Culture

Labyrinthula cells were inoculated, and suspension culture was performedby stirring at 25° C., 150 rpm in an Erlenmeyer flask or in a test tube.Subcultures were produced by adding a culture fluid to a new GY or PDliquid medium in a 1/200 to 1/10 volume after confirming proliferationfrom the logarithmic growth phase to the stationary phase. As required,the cell culture fluid was applied onto a PDA agar plate medium forconversion into an agar plate culture.

(4) Maintenance and Preservation Method of Labyrinthulomycetes

In addition to the subculture, cryopreservation was performed byproducing a glycerol stock. Specifically, glycerol (nacalai tesque) wasadded in a final concentration of 15% (v/v) to the logarithmic growthphase to stationary phase of a cell suspension in a GY liquid medium,and the cells were conserved in a −80° C. deep freezer.

Example 2

[Disruption of Thraustochytrium aureum C20 Elongase Gene]

[Example 2-1] Extraction of T. aureum ATCC 34304-Derived Total RNA, andmRNA Purification

A T. aureum ATCC 34304 culture fluid grown for 3 days using a GY liquidmedium was centrifuged at 3,500×g for 15 min, and the cells werecollected. After being suspended in sterilized physiological saline, thecells were washed by being recentrifuged. The cells were then rapidlyfrozen with liquid nitrogen, and ground into a powdery form with amortar. Total RNA was extracted from the resulting cell disruptionliquid, using Sepasol-RNA I Super (nacalai tesque). This was followed bypurification of mRNA from the total RNA using the Oligotex™-dT30 <Super>mRNA Purification Kit (Takara Bio) according to the manufacturer'sprotocol. The resulting total RNA and the mRNA were dissolved in asuitable amount of TE, and electrophoresed with a formalin-denatured gel(1% agarose/MOPS buffer). The result confirmed successful extraction ofthe total RNA, and purification of mRNA from the total RNA. It was alsoconfirmed that the RNA was not degraded by the RNase. In order tominimize RNA degradation, all experimental procedures were performedwith sanitary equipment such as rubber gloves and a mask. Allinstruments were RNase free, or were used after a diethylpyrocarbonate(nacalai tesque) treatment to deactivate the RNase. The solution used todissolve the RNA was prepared by adding the recombinant RNase inhibitorRNaseOUT™ (invitrogen) to sterilized Milli Q water treated withdiethylpyrocarbonate.

[Example 2-2] Isolation of T. aureum ATCC 34304-Derived Elongase Gene byRACE

Forward (elo-F;5′-TTY YTN CAY GTN TAY CAY CAY-3′) (SEQ ID NO: 1), andreverse (elo-R;5′-GCR TGR TGR TAN ACR TGN ARR AA-3′) (SEQ ID NO: 2)denatured oligonucleotides were synthesized, targeting the histidine box(His box) highly conserved in elongase genes. The oligonucleotides weresynthesized with a DNA synthesizer (Applied Biosystems). Then, byaddition of synthetic adapters to the 3′- and 5′-ends, 3′- and 5′-RACEcDNA libraries were produced by using the SMART™ RACE cDNA AmplificationKit (clontech) according to the manufacturer's protocol, respectively.By using these as templates, 3′- and 5′-RACE were performed using thesynthetic adapter-specific oligonucleotides, and the denaturedoligonucleotides elo-F and elo-R [PCR cycles: 94° C. 1 min/94° C. 30sec, 60° C. 30 sec, 72° C. 3 min, 30 cycles/72° C. 10 min/4° C. ∞]. Theresult confirmed bands for the specifically amplified 3′- and 5′-RACEproducts (FIG. 1). The total RACE product amounts were subjected toelectrophoresis with 1% agarose gel, and the isolated DNA fragments werecut out with a clean cutter or the like and extracted from the agarosegel according to the method described in Non-Patent Document 20. The DNAfragments were then TA cloned with a pGEM-T easy Vector (Promega), andthe base sequences were determined by the method of Sanger et al.(Non-Patent Document 21). Specifically, the base sequences weredetermined by using a dye terminator method, using a BigDyeR Terminatorv3.1 Cyele Sequencing Kit and a 3130 genetic analyzer (AppliedBiosystems) according to the manufacturers' protocols.

As a result, two sequences, 190 bp and 210 bp, named elo1 (SEQ ID NO: 3)and elo2 (SEQ ID NO: 4) were successfully identified for the 3′-RACEproduct, and one sequence, 200 bp, named elo3 (SEQ ID NO: 5) wassuccessfully identified for the 5′-RACE product. Because the elo1, elo2,and elo3 sequences had significant homology to the sequences of variouselongase genes, the results suggested that these sequences were partialsequences of the T. aureum ATCC 34304-derived elongase gene. In anattempt to obtain cDNA sequences by RACE, oligonucleotide primers wereredesigned for the elo1, elo2, and elo3. The oligonucleotide primersproduced are as follows.

elo1 forward oligonucleotide primer (SEQ ID NO: 6)(elo1-F1; 5′-TAT GAT CGC CAA GTA CGC CCC-3′) andreverse oligonucleotide primer (SEQ ID NO: 7)(elo1-R1; 5′-GAA CTG CGT CAT CTG CAG CGA-3′)elo2 forward oligonucleotide primer (SEQ ID NO: 8)(elo2-F1; 5′-TCT CGC CCT CGA CCA CCA AC-3′) andreverse oligonucleotide primer (SEQ ID NO: 9)(elo2-R1; 5′-CGG TGA CCG AGT TGA GGT AGC C-3′)elo3 forward oligonucleotide primer (SEQ ID NO: 10)(elo3-F1; 5′-CAA CCC TTT CGG CCT CAA CAA G-3′) andreverse oligonucleotide primer (SEQ ID NO: 11)(elo3-R1; 5′-TTC TTG AGG ATC ATC ATG AAC GTG TC-3′)

By using these forward and reverse oligonucleotide primers, RACE andbase sequence analysis of the amplification products were performed asabove. As a result, specifically amplified 3′- and 5′-RACE products wereobtained for elo1, and there was a complete match in the overlappingportion, identifying the sequence as a 1,139-bp elo1 cDNA sequence (SEQID NO: 12). Similarly, specifically amplified 3′- and 5′-RACE productswere obtained for elo3, and there was a complete match in theoverlapping portion, identifying the sequence as a 1,261-bp elo3 cDNAsequence (SEQ ID NO: 13).

It was found from the sequence analysis result that elo1 consisted of an825-bp translated region (SEQ ID NO: 15) coding for 275 amino acidresidues (SEQ ID NO: 14). It was also found from the result of a BLASTsearch that the sequence had significant homology to various elongasegenes, and completely coincided with the sequence of a known T.aureum-derived putative 45 elongase gene (NCBI accession No. C5486301).On the other hand, it was assumed that the elo3 consisted of a 951-bptranslated region (SEQ ID NO: 17) coding for 317 amino acid residues(SEQ ID NO: 16). It was also found from the result of a BLAST searchthat the sequence had significant homology to various elongase genes,and thus represented a T. aureum ATCC 34304-derived putative elongasegene. Note that the putative amino acid sequences of these genescontained His boxes highly conserved in elongase genes. From theseresults, elo1 and elo3 genes were identified as T. aureum ATCC34304-derived putative elongase genes, and were named TaELO1 and TaELO2,respectively,

[Example 2-3] TaELO1 and TaELO2 Phylogenetic Analysis

Elongases are broadly classified into three groups on the basis ofsubstrate specificity.

-   -   1. SFA/MUFA elongases (act on saturated fatty acids or        monovalent unsaturated fatty acids)    -   2. PUFA-elongases (single-step) (act on polyvalent unsaturated        fatty acids of certain chain lengths)    -   3. PUFA elongases (multi-step) (act on polyvalent unsaturated        fatty acids of various chain lengths)

According to the elongase phylogenetic analysis conducted by Meyer etal. (Non-Patent Document 22), there is a good correlation between thesubstrate specificity and the phylogenetic relationships.

Accordingly, a phylogenetic analysis was performed for TaELO1, TaELO2,and various other elongase genes derived from other organisms, using themethod of Meyer et al. Specifically, a molecular phylogenetic tree wascreated according to the neighbor-joining method (Non-Patent Document14), using the CLUSTAL Wprogram (Non-Patent Document 7). It was found asa result that the TaELO1 and TaELO2 were classified into thePUFA-elongases (single-step) group, suggesting that these elongases acton polyvalent unsaturated fatty acids of certain chain lengths (FIG. 2).

[Example 2-4] TaELO1 and TaELO2 Expression in Budding YeastSaccharomyces cerevisiae Host, and Fatty Acid Composition Analysis ofGene Introduced Strain

Expression vectors were constructed for TaELO1 and TaELO2 for theirexpression in budding yeast S. cerevisiae used as a host, as brieflydescribed below. A set of oligonucleotide primer (E1 HindIII; 5′-ATA AGCTTA AAA TGT CTA GCA ACA TGA GCG CGT GGG GC-3′) (SEQ ID NO: 18) and E1XbaI; 5′-TGT CTA GAA CGC GCG GAC GGT CGC GAA A-3′) (SEQ ID NO: 19) wasproduced using the sequence of the TaELO1 translated region. The E1HindIII is a forward oligonucleotide primer, and has a restrictionenzyme HindIII site (AAGCTT) at the 5′-end. The sequence in the vicinityof the TaELO1 start codon is modified by referring to a yeast consensussequence ((A/Y) A (A/U) AAUGUCU; the start codon is underlined)(Non-Patent Document 23). The E1 XbaI is a reverse oligonucleotideprimer, and has an XbaI site (TCTAGA) at the 5′-end.

In the same manner, a set of oligonucleotide primer (E2 HindIII; 5′-TAAAGC TTA AAA TGT CTA CGC GCA CCT CGA AGA GCG CTC C-3′) (SEQ ID NO: 20)and E2 XbaI; 5′-CAT CTA GAC TCG GAC TTG GTG GGG GCG CTT G-3′) (SEQ IDNO: 21) was produced using the sequence of the TaELO2 translated region.The E2 HindIII is a forward oligonucleotide primer, and has arestriction enzyme HindIII site at the 5′-end. The sequence in thevicinity of the TaELO2 start codon is modified by referring to a yeastconsensus sequence. The E2 XbaI is a reverse oligonucleotide primer, andhas an XbaI site at the 5′-end.

By using the two oligonucleotide primer sets, a PCR was performed usingthe 5′-RACE cDNA library of Example 2-2 as a template. The PCR amplifieda 949-bp TaELO1 translated region (SEQ ID NO: 22) and a 967-bp TaELO2translated region (SEQ ID NO: 23) having the restriction enzyme HindIIand the restriction enzyme XbaI site at the 5′-end and the 3′-end, andmodified to the yeast consensus sequence in the vicinity of the startcodon. Note that a PrimeSTARR DNA polymerase (Takara Bio) of highproofreading activity was used as the PCR enzyme to avoid extensionerrors [PCR cycles: 98° C. 2 min/98° C. 5 sec, 60° C. 5 sec, 72° C. 1.5min, 30 cycles/72° C. 7 min/4° C. cc].

After isolating the amplified PCR products with a 1% agarose gel, theDNA fragments were cut and extracted from the agarose gel. Aftertreatment with restriction enzymes HindIII and XbaI, the product waspurified again with an agarose gel. To construct a cyclic vector, theproduct was joined to a budding yeast expression vector pYES2/CT(invitrogen) with a DNA Ligation Kit <Mighty Mix> (Takara Bio) afterlinearizing the pYES2/CT vector with restriction enzymes HindIII andXbaI. This was followed by a base sequence analysis, which confirmedthat no PCR extension error occurred and no mutation was introduced tothe TaELO1 and TaELO2 translated region sequences introduced into thepYES2/CT. In this manner, a TaELO1 expression vector pYEELO1, and aTaELO2 expression vector pYEELO2 were successfully constructed.

The two expression vectors constructed above, and the pYES2/CT wereintroduced into the budding yeast S. cerevisiae by using the lithiumacetate technique according to the methods described in Non-PatentDocuments 15 and 16, and the transfectants were screened for. Theresulting transfectants (pYEELO1 introduced strain, pYEELO2 introducedstrain, and mock introduced strain) were cultured according to themethod of Qiu et al. (Non-Patent Document 24), and the cell-derivedfatty acids were extracted and methylesterificated. Note that eachculture was performed in a medium supplemented with α-linolenic acid(ALA, C18:349, 12, 15) and linoleic acid (LA, C18:249, 12) added as 49elongase substrates, stearidonic acid (STA, C18:446, 9, 12, 15) andγ-linolenic acid (GLA, C18:346, 9, 12) added as 46 elongase substrates,and eicosapentaenoic acid (EPA, C20:545, 8, 11, 14, 17) and arachidonicacid (AA, C20:445, 8, 11, 14) added as 45 elongase substrates. Here,each supplement was added in a final concentration of 0.2 mM. This wasfollowed by the gas chromatography (GC) analysis of themethylesterificated fatty acids according to the method of Abe et al.(Non-Patent Document 17). The GC analysis was performed with a gaschromatograph GC-2014 (Shimadzu Corporation) under the followingconditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

It was found as a result that, the pYEELO1 introduced strain had the 46elongase activity not found in the host (mock introduced strain),converting the stearidonic acid (STA) into eicosatetraenoic acid (ETA,C20:4Δ8, 11, 14, 17), and the γ-linolenic acid (GLA) intodihomo-γ-linolenic acid (DGLA, C20:3Δ8, 11, 14). The pYEELO1 introducedstrain also had the 49 elongase activity of converting the α-linolenicacid (ALA) into eicosatrienoic acid (ETrA, C20:3Δ11, 14, 17), and thelinoleic acid (LA) into eicosadienoic acid (EDA, C20:3Δ11, 14), and the45 elongase activity of converting the eicosapentaenoic acid (EPA) intoω3 docosapentaenoic acid (ω3 DPA, C22:5Δ7, 10, 13, 16, 19), and thearachidonic acid (AA) into docosatetraenoic acid (DTA, C22:4Δ7, 10, 13,16) (Table 1).

As for the pYEELO2 introduced strain, it was found that this strain hadthe Δ5 elongase activity not found in the host, converting EPA to ω3 DPA(C22:5Δ7, 10, 13, 16, 19), and AA to DTA. The pYEELO2 introduced strainalso had a weak 46 elongase activity, converting STA to ETA, and GLA toDGLA (Table 1). These results confirmed that the TaELO1 was a Δ6/Δ9/Δ5elongase, and the TaELO2 was a Δ5/Δ6elongase, contrary to the resultsexpected from the TaELO1 and TaELO2 substrate specificity in thephylogenetic analysis described in Example 2-3 and FIG. 2.

TABLE 1 mock TaELO1 TaELO2 LA addition (0.2 mM) LA 30.5 23.5 36.3 EDA0.2 8.9 0.2 Conversion 27.4 efficiency (%) GLA addition (0.2 mM) GLA44.0 7.6 43.6 DGLA 0.2 29.0 0.8 Conversion 79.3 1.9 efficiency (%) ARAaddition (0.2 mM) ARA 30.9 23.2 8.9 ADA — 5.8 13.6 Conversion 20.1 60.3efficiency (%) ALA addition (0.2 mM) ALA 49.1 25.8 47.1 ETrA 0.2 17.90.3 Conversion 41 efficiency (%) STA addition (0.2 mM) STA 46.2 8.3 40.5ETA 0.3 28.1 1.7 Conversion 77.2 4.0 efficiency (%) EPA addition (0.2mM) EPA 42.0 31.2 13.1 DPA 0.1 19.6 24.5 Conversion 25.3 65.1 efficiency(%) Conversion efficiency (%) = 100 × product (area)/substrate (area) +product (area) (n = 1)

[Example 2-5] Obtaining TaELO2 ORF Upstream and Downstream Regions byPCR Genome Walking

The TaELO2 ORF upstream and downstream regions as the homologousrecombination sites in a targeting vector for disrupting TaELO2 wereobtained by using the PCR genome walking technique, as briefly describedbelow.

T. aureum ATCC 34304 cell grown for 3 days using a GY liquid medium wasrapidly frozen with liquid nitrogen, and ground into a powdery form witha mortar. Then, genomic DNA was extracted according to the methoddescribed in Non-Patent Document 18, and dissolved in a suitable amountof TE. Genomic DNA levels and purity were assayed by O.D.260 and O.D.280measurements. This was followed by construction of a genomic DNA libraryby adding a cassette sequence with restriction enzyme sites to thegenomic DNA cut with various restriction enzymes, using a TaKaRa LA PCR™in vitro Cloning Kit (Takara Bio) according to the manufacturer'sprotocol. Then, by using the genomic DNA library as a template, a nestedPCR was performed according to the manufacturer's protocol, using theforward oligonucleotide primers E2 XbaI (Example 2-4; SEQ ID NO: 21) andelo3-F1 (Example 2-2; SEQ ID NO: 10) or the reverse oligonucleotideprimers E2 HindIII (Example 2-4; SEQ ID NO: 20) and elo3-R1 (Example2-2; SEQ ID NO: 11) produced from the TaELO2 sequence, and theoligonucleotide primers complementary to the cassette sequence (attachedto the kit). As a result, a 1,122-bp TaELO2 ORF upstream sequence (SEQID NO: 24), and a 1,204-bp TaELO2 ORF downstream sequence (SEQ ID NO:25) were successfully obtained.

[Example 2-6] Construction of TaELO2 Targeting Vector Using SelectionMarker Neor

A DNA fragment joining TaELO2 ORF upstream sequence/artificialNeor/TaELO2 ORF downstream sequence was produced by fusion PCR. Thefollowing oligonucleotide primers were used.

KO Pro F SmaI (SEQ ID NO: 26)(31 mer: 5′-CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T-3′) Pro R(SEQ ID NO: 27) (25 mer: 5′-GGT CGC GTT TAC AAA GCA GCG CAG C-3′) SNeo F(SEQ ID NO: 28) (52 mer; 5′-GCT GCG CTG CTT TGT AAA CGC GAC CATGAT TGA ACA GGA CGG CCT TCA CGC T-3′) SNeoR (SEQ ID NO: 29)(52 mer; 5′-TCG GGA GCC AGC CGG AAA CAG GTT CAAAAG AAC TCG TCC AGG AGG CGG TAG A-3′) Term F (SEQ ID NO: 30)(23 mer: 5′-ACC TGT TTC CGG CTG GCT CCC GA-3′) KO Term R SmaI(SEQ ID NO: 31) (27 mer: 5′-ATC CCG GGG CCG AGA ACG GGG TCG CCC-3′)

The oligonucleotide primers KO Pro F SmaI/Pro R were used for theamplification of the TaELO2 ORF upstream sequence using the T. aureumATCC 34304 genomic DNA of Example 2-5 as a template. The oligonucleotideprimers SNeo F/SNeo R were used for the amplification of the artificialNeor using artificial Neor as a template. The oligonucleotide primersTerm F/KO Term R SmaI were used for the amplification of the TaELO2 ORFdownstream sequence using the T. aureum ATCC 34304 genomic DNA ofExample 2-5 as a template. The PCR reaction was performed at a denaturetemperature of 98° C. for 10 seconds, and the annealing and theextension reaction were performed while appropriately adjusted accordingto the primer Tm and the amplification product length.

As a result, a 2, 696-bp sequence (SEQ ID NO: 32) joining TaELO2 ORFupstream sequence/artificial Neor/TaELO2 ORF downstream sequence wassuccessfully obtained, and the sequence after TA cloning with a pGEM-Teasy Vector (Promega) was used as a knockout vector, named pTKONeor.

[Example 2-7] Introduction of TKONeor into T. aureum ATCC 34304

The TaELO2 targeting vector pTKONeor using artificial Neor as aselection marker (Example 2-6) was used as a template, and the TaELO2ORF upstream sequence/artificial Neor/TaELO2 ORF downstream sequence wasamplified using a set of oligonucleotide primers KO Pro F SmaI (Example2-6, SEQ ID NO: 26)/KO Term R SmaI (Example 2-6, SEQ ID NO: 31), andPrimeSTAR HS DNA polymerase (Takara Bio) [PCR cycles: 98° C. 2 min/98°C. 10 sec, 68° C. 3 min, 30 cycles/68° C. 10 min/4° C. ∞]. The DNAfragments were extracted after electrophoresis using a 1% agarose gel,and dissolved in a suitable amount of TE after ethanol precipitation.The DNA fragment levels and the purity were assayed by O.D.260 andO.D.280 measurements. In the following, the DNA fragment will bereferred to as TKONeor.

This was followed by DNA penetration using the gene-gun technique.Specifically, T. aureum ATCC 34304 was cultured in a GY liquid mediumfrom the middle to late stage of the logarithmic growth phase at 25° C.,150 rpm, and the supernatant was removed by centrifugation at 3,500×g,4° C. for 10 min. The resulting cells were resuspended in a GY liquidmedium in 100 times the concentration of the original culture fluid, anda 20-μ1 portion of the cell suspension was evenly applied as a thinlayer of about a 3-cm diameter on a 5-cm diameter PDA agar plate mediumcontaining 1 mg/ml G418 (nacalai tesque). After drying, penetration wasperformed using a PDS-1000/He system (BioRad) under the followingconditions.

Target distance: 6 cm

Vacuum: 26 inches Hg

Micro carrier size: 0.6 μm

Rupture disk (penetration pressure): 1,100 psi

Thereafter, a PD liquid medium (100 μl) was dropped onto the PDA agarplate medium, and the cells were spread and statically cultured. As aresult, transfectants with the conferred G418 resistance were obtainedat the efficiency of 4.7×10¹ cfu/μg DNA.

[Example 2-8] PCR Using TKONeor-Introduced Transfectant Genomic DNA as aTemplate

Seven colonies of transfectants were collected with a toothpick, andinoculated in a GY liquid medium containing 0.5 mg/ml G418 (nacalaitesque). After multiple subculturing, genomic DNA was extracted from thecells using the method of Example 2-5, and dissolved in a suitableamount of TE after ethanol precipitation. The levels of extractedgenomic DNA and the purity were assayed by O.D.260 and O.D.280measurements. By using the genomic DNAs of the transfectants and thewild-type strain as templates, a PCR was performed with variousoligonucleotide primer sets. The following oligonucleotide primer setswere used.

(1) Neor detection: SNeoF (Example 2-6; SEQ ID NO: 28) and SNeoR(Example 2-6; SEQ ID NO: 29)

(2) KO verification 1: KO Pro F SmaI (Example 2-6; SEQ ID NO: 26) and KOTerm R SmaI (Example 2-6; SEQ ID NO: 31)

(3) KO verification 2: E2 KO ProF EcoRV (30 mer: 5′-GGA TAT CCC CCG CGAGGC GAT GGC TGC TCC-3′) (SEQ ID NO: 33) and SNeoR

(4) KO verification 3: SNeoF and E2 KO Term R EcoRV (30 mer: 5′-TGA TATCGG GCC GCG CCC TGG GCC GTA GAT-3′) (SEQ ID NO: 34)

(5) TaELO2 amplification: E2 HindIII (Example 2-4; SEQ ID NO: 20) and E2XbaI (Example 2-4; SEQ ID NO:21) (FIG. 3A)

Six out of the seven clones analyzed were transfectants by randomintegration, and the homologous recombination replacement of TaELO2 ORFwith Neor was confirmed in the remaining clone (FIG. 3B, lanes 9 and13). It was also found that this was accompanied by the simultaneousTaELO2 ORF amplification (FIG. 3B, lane 17). These results suggested thepossibility that the T. aureum ATCC 34304 was a diploid or higherploidy, or the TaELO2 was a multicopy gene.

[Example 2-9] Confirmation of TaELO2 Copy Number by Southern Blotting

The following experiments were conducted according to the methodsdescribed in DIG Application Manual [Japanese version] 8th, RocheApplied Science (Non-Patent Document 25). Specifically, the genomic DNAof the wild-type strain was cut with various restriction enzymes, andelectrophoresed in 2.5 μg per lane using a 0.7% SeaKemR GTGR agarose(Takara Bio). This was transferred to a nylon membrane (Hybond™-N+, GEHealthcare), and hybridized at 48° C. for 16 hours with DIG-labeledprobes produced by using a PCR DIG Probe Synthesis Kit (Roche AppliedScience). The following oligonucleotide primer set was used for theproduction of the DIG-labeled probes.

TaELO2 det F (SEQ ID NO: 35)(25 mer: 5′-GTA CGT GCT CGG TGT GAT GCT GCT C-3′) TaELO2 det R(SEQ ID NO: 36) (24 mer: 5′-GCG GCG TCC GAA CAG GTA GAG CAT-3′)[PCR cycles: 98° C. 2 min/98° C. 30 sec, 65° C. 30 sec,72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]

Detection of the hybridized probes was made by using a chromogenicmethod (NBT/BCIP solution).

As a result, a single band was detected in all lanes treated with thevarious restriction enzymes (FIG. 4), suggesting that the TaELO2 was asingle copy gene. The result thus suggested that the T. aureum ATCC34304 was a diploid or higher ploidy.

[Example 2-10] Evaluation of TKONeor-Introduced Transfectants bySouthern Blotting

Southern blotting was performed by using the method of Example 2-9.Specifically, the genomic DNAs of the wild-type strain and thetransfectants digested with EcoRV and PstI were subjected to southernblotting using a chromogenic method (NBT/BCIP solution), usingDIG-labeled probes PCR amplified with a set of oligonucleotide primersuprobe F (35 mer: 5′-ATC CGC GTA TAT ATC CGT AAA CAA CGG AAC ATT CT-3′)(SEQ ID NO: 37) and uprobe R (26 mer: 5′-CTT CGG GTG GAT CAG CGA GCG ACAGC-3′) (SEQ ID NO: 38) [PCR cycles: 98° C. 2 min/98° C. 30 sec, 65° C.30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. Here, in contrastto about a 1.2-kbp DNA fragment detected for the wild-type allele, abouta 2.5-kbp DNA fragment was detected for the mutant allele that underwentthe homologous recombination replacement of TaELO2 ORF with Neor (FIG.5A).

Because the wild-type allele band was simultaneously detected with themutant allele band in the transfectants (FIG. 5B), the analysis resultsuggested that the T. aureum ATCC 34304 was a diploid or higher ploidy.

[Example 2-11] Construction of TaELO2 Targeting Vector Using SelectionMarker Hygr

A TaELO2 targeting vector was constructed with a selection marker Hygrto disrupt the remaining wild-type allele.

First, a fusion PCR was performed to join Hygr to a T. aureum ATCC34304-derived ubiquitin promoter sequence. The following oligonucleotideprimers were used.

ubi-600p F (SEQ ID NO: 39) (27 mer: 5′-GCC GCA GCG CCT GGT GCA CCC GCCGGG-3′) ubi-hygro R (SEQ ID NO: 40)(59 mer: 5′-TCG CGGG TGA GTT CAG GCT TTT TCA TGTTGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3′) ubi-hygro F(SEQ ID NO: 41) (57 mer; 5′-AGC GAC CTA AGC AAC ACT AGGC CAA CATGAA AAA GCC TGA ACT CAC CGC GAC GTC TG-3′) hygro R (SEQ ID NO: 42)(29 mer; 5′-CTA TTC CTT TGC CCT CGG ACG AGT GCT GG-3′)

The oligonucleotide primers ubi-600p F/ubi-hygro R were used for theamplification of the T. aureum ATCC 34304-derived ubiquitin promotersequence using the T. aureum ATCC 34304 genomic DNA of Example 2-5 as atemplate. The oligonucleotide primers ubi-hygro F/hygro R were used forthe amplification of the artificial Hygr using pcDNA 3.1 Zeo (Invitogen)as a template. The PCR reaction was performed at a denature temperatureof 98° C. for 10 seconds, and the annealing and the extension reactionwere appropriately adjusted according to the primer Tm and theamplification product length.

As a result, a 1,636-bp (SEQ ID NO: 43) joining T. aureum ATCC34304-derived ubiquitin promoter sequence/Hygr was successfullyobtained, and the sequence after TA cloning with a pGEM-T easy Vector(Promega) was named pTub600Hygr.

By using the pTub600Hygr as a template, a PCR was performed withPrimeSTAR HS DNA polymerase (Takara Bio) to prepare a T. aureum ATCC34304-derived ubiquitin promoter sequence/Hygr DNA fragment containingNheI and XbaI sites added to the 5′ end and the 3′ end, respectively.The PCR was run under the following conditions using a set ofoligonucleotide primers ubi-600 pF NheI (33 mer: 5′-GTG CTA GCC GCA GCGCCT GGT GCA CCC GCC GGG-3′) (SEQ ID NO: 44) and hygro R XbaI (37 mer:5′-GTT CTA GAC TAT TCC TTT GCC CTC GGA CGA GTG CTG G-3′) (SEQ ID NO: 45)[PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3 min, 30 cycles/68° C.10 min/4° C. co]. Separately, by using the pTKONeor of Example 2-6 as atemplate, a PCR was performed with PrimeSTAR HS DNA polymerase (TakaraBio) to prepare a linear vector that did not contain the Neor of thepTKONeor of Experiment Example 2-6 and to which NheI and XbaI sites wereadded to the 3′ end and the 5′ end, respectively. The PCR was run underthe following conditions using a set of oligonucleotide primers KO vec FXbaI (37 mer: 5′-GTT CTA GAC CTG TTT CCG GCT GGC TCC CGA GCC ATG C-3′)(SEQ ID NO: 46) and KO vec R NheI (40 mer: 5′-GTG CTA GCG GTC GCG TTTACA AAG CAG CGC AGC AAC AGA A-3′) (SEQ ID NO: 47) [PCR cycles: 98° C. 2min/98° C. 10 sec, 68° C. 3 min, 30 cycles/68° C. 10 min/4° C. ∞]. Theboth DNA fragments were digested with restriction enzymes NheI and XbaI,and purified with an agarose gel to construct a cyclic vector using aLigation Convenience Kit (Nippon Gene).

The TaELO2 targeting vector using Hygr as a selection marker thusconstructed used the pGEM-T easy Vector (Promega) as the platform, andcontained a 3,537-bp insert sequence (SEQ ID NO: 48) of TaELO2 ORFupstream sequence/T. aureum ATCC 34304-derived ubiquitin promotersequence/Hygr/TaELO2 ORF downstream sequence. This was namedpTKOub600Hygr.

[Example 2-12] Reintroduction of KOub600Hygr, and Evaluation ofTransfectants by PCR Using Genomic DNA as Template, and by SouthernBlotting and RT-PCR

The constructed TaELO2 targeting vector pTKOub600Hygr (Example 2-11)using Hygr as a selection marker was used as a template, and the TaELO2ORF upstream sequence/T. aureum ATCC 34304-derived ubiquitin promotersequence/Hygr/TaELO2 ORF downstream sequence was amplified with aPrimeSTAR HS DNA polymerase (TakaraBio), using a set of oligonucleotideprimers KO Pro F SmaI (Example 2-6, SEQ ID NO: 26)/KO Term R SmaI(Example 2-8, SEQ ID NO: 31) [PCR cycles: 98° C. 2 min/98° C. 10 sec,68° C. 3.5 min, 30 cycles/68° C. 10 min/4° C. ∞]. The resulting DNAfragment was named KOub600Hygr. This was introduced to the transfectantsobtained in Example 2-6 by using the technique described therein, andstatically cultured on a 1 mg/ml G418 (nacalai tesque)-containing PDAagar plate medium for 24 hours. The cells were collected, and staticallycultured on a PDA agar plate medium supplemented with 1 mg/ml G418(nacalai tesque) and 2 mg/ml hygromycin B (Wako Pure ChemicalIndustries, Ltd.). As a result, large numbers of transfectants wereobtained (introduction efficiency: 1.02×10³ cfu/μg DNA).

Fifty clones were collected, and subcultured multiple times in a GYliquid medium supplemented with 1 mg/ml G418 (nacalai tesque) and 2mg/ml hygromycin B (Wako Pure Chemical Industries, Ltd.). Then, genomicDNA was extracted by using the same technique used in Example 2-5, anddissolved in a suitable amount of TE after ethanol precipitation. Thelevels of extracted genomic DNA and the purity were assayed by O.D.260and O.D.280 measurements. By using the genomic DNAs of the resultingtransfectants and the wild-type strain as templates, a PCR was performedwith various oligonucleotide primer sets [PCR cycles: 98° C. 2 min/98°C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 10 min/4° C. ∞]. The followingoligonucleotide primer sets were used.

(1) TaELO2 ORF detection: SNeoF (Example 2-6; SEQ ID NO: 28) and SNeoR(Example 2-6; SEQ ID NO: 29)

(2) KO verification: E2 KO Pro F EcoRV (Example 2-8; SEQ ID NO: 33) andubi-hygro R (Example 2-11; SEQ ID NO: 40) (FIG. 6A).

It was suggested that 14 out of the 50 clones analyzed weretransfectants that underwent homologous recombination through TaELO2 ORFreplacement (FIG. 6B, arrow). It was also confirmed that the TaELO2 ORFwas not amplified in these clones (FIG. 6C).

This was followed by southern blotting using the same technique used inExample 2-10. Specifically, the genomic DNAs of the wild-type strain andthe transfectants digested with EcoRV and PstI were subjected tosouthern blotting using a chromogenic method (NBT/BCIP solution), usingDIG-labeled probes prepared with a set of oligonucleotide primers uprobeF (SEQ ID NO: 37) and uprobe R (SEQ ID NO: 38). Here, about a 1.2-kbpDNA fragment was detected for the wild-type allele. In contrast, about a2.5-kbp DNA fragment was detected for the mutant allele that underwentthe homologous recombination replacement of TaELO2 ORF with Neor, andabout a 1.9-kbp DNA fragment was detected for the mutant allele thatunderwent the homologous recombination replacement of TaELO2 ORF withHygr (FIG. 7A).

The analysis revealed that the wild-type allele band of about a 2.5 kbpwas absent in the resulting transfectants, and a new band, about 1.9kbp, was detected for the mutant allele in which the TaELO2 ORF wasreplaced with Hygr (FIG. 7B).

Southern blotting using a chromogenic method (NBT/BCIP solution) wasalso performed for the genomic DNAs of the wild-type strain and thetransfectants (clones 1, 8, 9, and 10) digested with EcoRV, usingTaELO2-detecting DIG-labeled probes prepared by PCR using a set ofoligonucleotide primers TaELO2 probe F (30 mer: 5′-ATG GCG ACG CGC ACCTCG AAG AGC GCT CCG-3′) (SEQ ID NO: 49) and TaELO2 probe R (30 mer:5′-AGG ATC ATC ATG AAC GTG TCG CTC CAG TCG-3′) (SEQ ID NO: 50) [PCRcycles: 98° C. 2 min/98° C. 30 sec, 65° C. 30 sec, 72° C. 1 min, 30cycles/72° C. 7 min/4° C. ∞]. Here, TaELO2 was detected as about a2.5-kbp DNA fragment (FIG. 7A).

The analysis revealed that in contrast to the wild-type strain in whichthe TaELO2 was detected (FIG. 8, lane 1), TaELO2 was not detected in anyof the transfectants (FIG. 8, lanes 2 to 5).

To examine the TaELO2 disruption at the mRNA level, TaELO2 mRNAdetection was performed by RT-PCR. Total RNA was extracted from thecells of the wild-type strain and the transfectants (clones 1, 8, 9, and10) cultured for 3 days in GY liquid media, using Sepasol-RNA I Super(nacalai tesque) as in Example 2-1. The total RNA (50 μg) was cleaned upusing an RNeasyMini Kit (QIAGEN) according to the manufacturer'sprotocol, and treated at 37° C. for 1 hour using 50 U Recombinant DNaseI (Takara Bio) to degrade and remove the contaminated genomic DNA. Byusing the resulting total RNA as a template, a single-stranded cDNAlibrary was created using oligo (dT) primer (Novagen) and PrimeScriptReverse Transcriptase (Takara Bio) according to the manufacturers'protocols. By using the resulting single-stranded cDNA library as atemplate, the TaELO2 ORF was amplified with a set of oligonucleotideprimers E2 HindIII (Example 2-4; SEQ ID NO: 20) and E2 XbaI (Example2-4; SEQ ID NO:21), and LA taq Hot Start Version (Takara Bio) [PCRcycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 10min/4° C. ∞].

It was found as a result that the TaELO2 mRNA detected in the wild-typestrain (FIG. 9, lane 5) was not detected in any of the transfectants(clones 1, 8, 9, and 10) (FIG. 9, lanes 1 to 4).

As demonstrated above, TaELO2-deficient homozygotes with the completedisruption of TaELO2 were successfully obtained. It was also found thatthe T. aureum ATCC 34304 was a diploid.

[Example 2-13] Comparison of Fatty Acid Compositions of Wild-Type Strainand TaELO2-Deficient Homozygote

The fatty acid compositions of the TaELO2-deficient homozygote and thewild-type strain of Example 2-12 were compared by the GC analysis ofmethylesterificated fatty acids. Specifically, the cells of theTaELO2-deficient homozygotes and the wild-type strain cultured for 5days in GY liquid media were collected, and the fatty acids from thesecells were extracted and methylesterificated by using the methodsdescribed in Example 2-4, and subjected to GC analysis. The GC analysiswas performed with a gas chromatograph GC-2014 (Shimadzu Corporation)under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

As a result, the level of the EPA as a TaELO2 substrate showed about atwo-fold increase in the TaELO2-deficient homozygote compared to thewild-type strain, whereas the level of the downstream metabolite DHA waslower than in the wild-type strain (FIG. 10).

The present invention is the first example of the modification of fattyacid compositions through disruption of genes that form thedesaturase/elongase pathways in Labyrinthula. Specifically, the presentinvention has elucidated the involvement of the desaturase/elongasepathways in the PUFA biosynthesis in the Labyrinthula T. aureum, andshows that modification of fatty acid composition is possible byknocking out the constitutive genes. In the future, it would be possibleto perform molecular breeding of Labyrinthulomycetes that selectivelyproduce industrially useful PUFAs in large quantities in a PUFAbiosynthetic pathway artificially created from combinations of geneticmodifications such as overexpression of foreign desaturase/elongasegenes, and PUFA-PKS gene knockouts.

Example 3

[Disruption of Parietichytrium sarkarianum C20 Elongase Gene]

[Example 3-1] Subcloning of SV40 Terminator Sequence

An SV40 terminator sequence was amplified with PrimeSTAR HS DNApolymerase (Takara Bio), using a pcDNA 3.1 Myc-His vector as a template.The PCR primers used are as follows. RHO58 was set on the SV40terminator sequence, and contains BglII and BamHI linker sequences.RHO52 was set on the SV40 terminator sequence, and contains a BglIIsequence [RHO58: 34mer: 5′-CAG ATC TGG ATC CGC GAA ATG ACC GAC CAA GCGA-3′ (SEQ ID NO: 51), RHO52: 24mer: 5′-ACG CAA TTA ATG TGA GAT CTAGCT-3′ (SEQ ID NO: 52)]. The sequence was cloned into a pGEM-T easyvector (Promega) after being amplified under the following conditions[PCR cycles: 98° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 30cycles/72° C. 1 min]. The sequence was confirmed with a Dye TerminatorCycle Sequencing Kit (BECKMAN COULTER) after being amplified withEscherichia coli, and was named pRH27.

A plasmid (pRH27) containing the subcloned SV40 terminator sequence (342bp, SEQ ID NO: 53) is shown in FIG. 11.

[Example 3-2] Production of Artificial Neomycin-Resistant Gene Cassette

The Thraustochytrium aureum ATCC 34304 strain was cultured in GY medium,and cells at the late stage of the logarithmic growth phase werecentrifuged at 4° C., 3,500×g for 5 min to obtain a pellet. The pelletwas then disrupted after being frozen with liquid nitrogen. The celldisruption liquid was extracted with phenol, and precipitated withethanol. The precipitate was then dissolved in a TE solution. Thenucleic acids dissolved in the TE solution were treated with RNase at37° C. for 30 min to degrade the RNA, and extracted again with phenol.After ethanol precipitation, the precipitate was dissolved in a TEsolution. The DNA concentration was calculated by measuring A260/280.

By using this as a template, a ubiquitin promoter sequence (619 bp, SEQID NO: 54) was amplified using a PrimeSTAR HS DNA polymerase with GCBuffer (Takara Bio). The PCR primers used are as follows. RHO53 was seton the ubiquitin promoter sequence, and contains a BglII linkersequence. The TKO′ contains the ubiquitin promoter sequence and anartificial neomycin-resistant gene sequence [RHO53: 36mer: 5′-CCC AGATCT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′ (SEQ ID NO: 55), TKO′: 58mer:5′-CGT GAA GGC CGT CCT GTT CAA TCA TGT TGG CTA GTG TTG CTT AGG TCG CTTGCT GCT G-3′ (SEQ ID NO: 56)] [PCR cycles: 98° C. 2 min/98° C. 10 sec,68° C. 1 min, 30 cycles/68° C. 1 min].

An artificial neomycin-resistant gene sequence (826 bp, SEQ ID NO: 57)was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer(TakaraBio), using the artificial neomycin-resistant gene sequence as atemplate. The PCR primers used are as follows. TKO2 contains theubiquitin promoter sequence and the artificial neomycin-resistant genesequence. RHO57 contains the artificial neomycin-resistant genesequence, and has a BglII linker sequence [TKO2: 54mer: 5′-AGC GAC CTAAGC AAC ACT AGC CAA CAT GAT TGA ACA GGA CGG CCT TCA CGC TGG-3′ (SEQ IDNO: 58), RHO57: 26mer: 5′-CAG ATC TCA AAA GAA CTC GTC CAG GA-3′ (SEQ IDNO: 59)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30cycles/68° C. 1 min].

By using SEQ ID NOS: 54 and 57 as templates, a fusion PCR was performedwith RHO53 (SEQ ID NO: 55) and RHO57 (SEQ ID NO: 59) according to themethod described in Non-Patent Document 19. The product was amplifiedunder the following conditions by using an LA taq Hot start version(Takara Bio) as an enzyme, and digested with BglII [PCR cycles: 94° C. 2min/94° C. 20 sec, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68° C. 1 min(1° C./10 sec from 55° C. to 68° C.) (FIG. 12).

The fused product Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter-artificial neomycin-resistant gene sequence (1,395 bp, SEQ IDNO: 60) was digested with BglII, and ligated to the BamHI site of thepRH27 of Example 3-1. The resulting plasmid was amplified withEscherichia coli, and the sequence was confirmed by using a DyeTerminator Cycle Sequencing Kit (BECKMAN COULTER) and named pRH31.

The product artificial neomycin-resistant gene cassette (pRH31) is shownin FIG. 13.

[Example 3-3] Production of Hygromycin-Resistant Gene Cassette

By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as atemplate, a ubiquitin promoter sequence (617 bp, SEQ ID NO: 61) wasamplified with a PrimeSTAR HS DNA polymerase with GC Buffer (TakaraBio). The PCR primers used are as follows. RHO53 was set on theubiquitin promoter sequence, and contains a BglII linker sequence. KSO8contains the ubiquitin promoter sequence and a hygromycin-resistant genesequence [RHO53: 36mer: 5′-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCCGGG-3′ (Example 3-2; SEQ ID NO: 55), KSO8: 58mer: 5′-TCG CGG TGA GTT CAGGCT TTT TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3′ (SEQ ID NO:62)] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 68° C. 2 min, 30cycles/68° C. 2 min].

By using a pcDNA 3.1/Hygro (invitrogen) as a template, ahygromycin-resistant gene (1,058 bp, SEQ ID NO: 63) was amplified with aPrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio). The PCR primersused are as follows. KSO7 contains the ubiquitin promoter sequence andthe hygromycin-resistant gene sequence. RHO56 contains thehygromycin-resistant gene, and has a BglII linker sequence [KSO7: 56mer:5′-AGC GAC CIA AGC AAC ACT AGC CAA CAT GAA AAA GCC TGA ACT CAC CGC GACGTC TG-3′ (SEQ ID NO: 64), RHO56: 36mer: 5′-CAG ATC TCT ATT CCT TTG CCCTCG GAC GAG TGC TGG-3′ (SEQ ID NO: 65)] [PCR cycles: 98° C. 2 min/98° C.30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min].

By using SEQ ID NOS: 61 and 63 as templates, a fusion PCR was performedwith RHO53 (Example 3-2; SEQ ID NO: 55) and RHO56 (SEQ ID NO: 65)according to the method described in Non-Patent Document 19. The productwas amplified under the following conditions using an LA taq Hot startversion (Takara Bio) as an enzyme, and digested with BglII [PCR cycles:94° C. 2 min/94° C. 20 sec, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68°C. 1 min (1° C./10 sec from 55° C. to 68° C.)] (FIG. 14).

The fused product Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter-pcDNA 3.1/Hygro (invitrogen)-derived hygromycin-resistant gene(1,625 bp, SEQ ID NO: 66) was digested with BglII, and ligated to theBamHI site of the pRH27 of Example 3-1 (FIG. 11). The resulting plasmidwas amplified with Escherichia coli, and the sequence was confirmed byusing a Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This wasnamed pRH32.

The product artificial neomycin-resistant gene cassette (pRH32) is shownin FIG. 15.

[Example 3-4] Cloning of Parietichytrium C20 Elongase Gene

The Parietichytrium sarkarianum SEK364 genomic DNA extracted by usingthe method of Example 3-2 was extracted, and the genome was decoded.

A forward oligonucleotide (PsTaELO2 F1; 5′-CCT TCG GCG CTC CTC TTA TGTATG T-3′) (SEQ ID NO: 67) and a reverse oligonucleotide (PsTaELO2 R2;5′-CAA TGC AAG AGG CGA ACT GGG AGA G-3′) (SEQ ID NO: 68) weresynthesized by targeting a conserved region in the C20 elongase gene.The oligonucleotides PsTaELO2 F1 and PsTaELO2 R2 were then used for aPCR performed with an LA taq Hot start version (TaKaRa) using theParietichytrium sarkarianum SEK364 genomic DNA prepared by using themethod of Example 3-2 as a template [PCR cycles: 98° C. 1 min/98° C. 10sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. Theresulting specific amplification product was gel purified, and the basesequence was analyzed by direct sequencing. The sequence showedsignificant homology with the sequence of a known C20 elongase gene,suggesting that the sequence was a partial sequence of theParietichytrium sarkarianum SEK364-derived C20 elongase gene.

This was followed by cloning of the Parietichytrium sarkarianumSEK364-derived C20 elongase gene by 3′- and 5′-RACE, as in Example 2-2.First, forward oligonucleotide primers (PsRACE F1; 5′-TGG GGC TCT GGAACC GCT GCT TAC G-3′) (SEQ ID NO: 69) and (PsRACE F2; 5′-CTT CCA GCT CTCCCA GTT CGC CTC T-3′) (SEQ ID NO: 70), and reverse oligonucleotideprimers (PsRACE R1; 5′-CGG GTT GTT GAT GTT GAG CGA GGT G-3′) (SEQ ID NO:71) and (PsRACE R2; 5′-CCC ACG CCA TCC ACG AGC ACA CCA C-3′) (SEQ ID NO:72) were designed. This was followed by 3′- and 5′-RACE using asynthetic adapter-specific oligonucleotide and the oligonucleotidePsRACE F1 or PsRACE R1, using the cDNA library created with the SMART™RACE cDNA Amplification Kit (Clontech) as a template [PCR cycles: 94° C.30 sec 5 cycles/94° C. 30 sec, 70° C. 30 sec, 72° C. 3 min, 5 cycles/94°C. 30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/4° C. ∞]. By using theresulting both RACE products as templates, a nested PCR was performedusing a synthetic adapter-specific oligonucleotide, and theoligonucleotide PsRACE F2 or PsRACE R2 [PCR cycles: 94° C. 1 min/94° C.30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/72° C. 10 min/4° C. ∞].The resulting specific product was gel purified, and the base sequencewas analyzed after being TA cloned with a pGEM-T easy Vector (Promega).The result confirmed that the product was a Parietichytrium sarkarianumSEK364-derived C20 elongase gene.

A sequence (957 bp, SEQ ID NO: 73) containing the C20 elongase genesequence was amplified with an LA taq Hot start version (Takara Bio),using the Parietichytrium genomic DNA extracted by using the method ofExample 3-2 as a template. The PCR primers used are as follows. RHO153contains a start codon, and has a BamHI site as a linker sequence.RHO154 contains a stop codon, and has a BamHI site as a linker sequence[RHO153: 32 mer: 5′-CCC GGA TCC ATG GCA GCT CGC GTG GAG AAA CA-3′ (SEQID NO: 74), RHO154: 33 mer: 5′-CCC GGA TCC TTA CTG AGC CTT CTT GGA GGTCTC-3′ (SEQ ID NO: 75)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C.1 min, 30 cycles/68° C. 2 min].

The resulting DNA fragment was cloned into a pGEM-T easy vector, andamplified with Escherichia coli. Then, the sequence was confirmed with aDye Terminator Cycle Sequencing Kit (BECKMAN COULTER).

The 936-bp Parietichytrium C20 elongase gene (SEQ ID NO: 76) was cloned,and named pRH80 (FIG. 16). The amino acid sequence is represented by SEQID NO: 77.

[Example 3-5] Production of Base Plasmid for Parietichytrium C20Elongase Gene Targeting Vector Production

By using the pRH80 produced in Example 3-4 (FIG. 16) as a template,amplification was performed with a PrimeSTAR Max DNA Polymerase (TakaraBio), using a primer set of the reverse orientation prepared for theinsertion of the BglII site in a portion halfway along the C20 elongasegene sequence. The PCR primers used were as follows, and the bothprimers have BglII linker sequences [RHO155: 26 mer: 5′-ACA AAG ATC TCGACT GGA CCG ACA CC-3′ (SEQ ID NO: 78), RHO156: 27 mer: 5′-AGT CGA GATCTT TGT CAG GAG GTG GAC-3′ (SEQ ID NO: 79)] [PCR cycles: 98° C. 2min/98° C. 10 sec, 56° C. 15 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].After the amplification under these conditions, the product was digestedwith BglII, and allowed to self-ligate. The ligated sample was amplifiedwith Escherichia coli, and the sequence was confirmed by using a DyeTerminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH83.The 935-bp C20 elongase gene sequence with the inserted BglII site isrepresented by SEQ ID NO: 80.

The produced base plasmid (pRH83) for Parietichytrium C20 elongase genetargeting vector production is shown in FIG. 17.

[Example 3-6] Production of Targeting Vectors (ArtificialNeomycin-Resistant Gene and Hygromycin-Resistant Gene)

The pRH31 (FIG. 13) of Example 3-2 was digested with BglII, and a DNAfragment containing an artificial neomycin-resistant gene cassette wasligated to the BglII site of the pRH83 (FIG. 17) of Example 3-5. Thiswas named pRH85.

The pRH32 (FIG. 15) of Example 3-3 was digested with BglII, and a DNAfragment containing a hygromycin-resistant gene cassette was ligated tothe BglII site of the pRH83 (FIG. 17) of Example 3-5. This was namedpRH86.

The two targeting vectors (pRH85 and 86) produced are shown in FIG. 18.

[Example 3-7] Introduction of C20 Elongase Gene Targeting Vector

By using the two targeting vectors produced in Example 3-6 as templates,the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio),using the RHO153 (Example 3-4; SEQ ID NO: 74) and RHO154 (Example 3-4;SEQ ID NO: 75) as primers [PCR cycles: 98° C. 2 min/98° C. 30 sec, 68°C. 2 min, 30 cycles/68° C. 2 min]. After being extracted withphenol-chloroform and then with chloroform, the DNA was precipitatedwith ethanol, and the precipitate was dissolved in 0.1×TE. The DNAconcentration was then calculated by measuring A260/280. The introducedfragment obtained from using the pRH85 (FIG. 18) of Example 3-6 as atemplate was 2,661 bp, and had the following sequence order: First halfof Parietichytrium C20 elongase gene-SV40 terminator sequence-artificialneomycin-resistant gene sequence-ubiquitin promoter sequence-second halfof Parietichytrium C20 elongase gene (SEQ ID NO: 81). The introducedfragment obtained from using the pRH86 (FIG. 18) of Example 3-6 as atemplate was 2,892 bp, and had the following sequence order: First halfof Parietichytrium C20 elongase gene-SV40 terminatorsequence-hygromycin-resistant gene sequence-ubiquitin promotersequence-second half of Parietichytrium C20 elongase gene (SEQ ID NO:82).

The Parietichytrium sarkarianum SEK364 strain was cultured in a GYmedium for 4 days, and cells in the logarithmic growth phase were usedfor gene introduction. The DNA fragment (0.625 μg) was then introducedinto cells corresponding to OD600=1 to 1.5 using the gene-gun technique(microcarrier: 0.6-micron gold particles, target distance: 6 cm, chambervacuum: 26 mmHg, rupture disk: 1,550 PSI). After a 24-hour recoverytime, the cells with the introduced gene were applied onto a PDA agarplate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin). As aresult, 10 to 20 drug resistant strains were obtained per penetration.

[Example 3-8] Identification of C20 Elongase Gene Gene TargetingHomologous Recombinant

Genomic DNA was extracted from the Parietichytrium sarkarianum SEK364strain, the C20 elongase gene hetero homologous recombinant, and the C20elongase gene homo homologous recombinant (gene disrupted strain) byusing the method described in Example 3-2, and the DNA concentration wascalculated by measuring A260/280. By using this as a template, a PCR wasperformed with an LA taq Hot start version (Takara Bio) to confirm thegenome structure. The positions of the primers, combinations used forthe amplification, and the expected sizes of the amplification productsare shown in FIG. 19. RHO184 and RHO185 were set on the upstream anddownstream sides, respectively, of the C20 elongase. RHO142 and RHO143were set on the artificial neomycin-resistant gene. RHO140 and RHO141were set on the hygromycin-resistant gene [RHO140: 20 mer: 5′-GGT TGACGG CAA TTT CGA TG-3′ (SEQ ID NO: 83), RHO141: 22 mer: 5′-CCT CCT ACATCG AAG CTG AAA G-3′ (SEQ ID NO: 84), RHO142: 21 mer: 5′-CTT CTC GGG CTTTAT CGA CTG-3′ (SEQ ID NO: 85), RHO143: 22 mer: 5′-TAA GGT CGG TCT TGACAA ACA G-3′ (SEQ ID NO: 86), RHO184: 24 mer: 5′-AGT AGT CCC CGA TTT GGTAGT TGA-3′ (SEQ ID NO: 87), RHO185: 22 mer: 5′-GGC AGA GAG CAA AAA CACGAG C-3′ (SEQ ID NO: 88)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68°C. 4 min, 30 cycles/68° C. 7 min].

C20 elongase knockout strains were obtained that showed no amplificationof the wild-type allele (Wt allele) and the artificialneomycin-resistant gene allele (NeoR allele) and thehygromycin-resistant gene allele (HygR allele) (FIG. 20).

[Example 3-9] Changes in Fatty Acid Composition by C20 ElongaseDisruption

Parietichytrium sarkarianum SEK364, and the gene disrupted strains werecultured in GY media. Cells from the late stage of the logarithmicgrowth phase were centrifuged at 4° C., 3,000 rpm for 10 min to form apellet, suspended in 0.9% NaCl, and washed. The cells were furthercentrifuged at 4° C., 3,000 rpm for 10 min, and the pellet was suspendedin sterile water, and washed. After further centrifugation at 3,000 rpmfor 10 min, the cells were freeze dried after removing the supernatant.

Then, 2 ml of methanolic KOH (7.5% KOH in 95% methanol) was added to thefreeze dried cells, and, after being vortexed, the cells wereultrasonically disrupted (80° C., 30 min). The cells were vortexed afteradding sterile water (500 μl), and vortexed again after adding n-hexane(2 ml). This was followed by centrifugation at 3,000 rpm for 10 min, andthe upper layer was discarded. The cells were vortexed again afteradding n-hexane (2 ml), and centrifuged at 3,000 rpm for 10 min. Afterdiscarding the upper layer, 6 N HCl (1 ml) was added to the remaininglower layer, and the mixture was vortexed. The mixture was vortexedagain after adding n-hexane (2 ml). This was followed by centrifugationat 3,000 rpm for 10 min, and the upper layer was collected. The mixturewas further vortexed after adding n-hexane (2 ml), centrifuged at 3,000rpm for 10 min, and the upper layer was collected. The collected upperlayer was then concentrated and dried with nitrogen gas. Theconcentrated dry sample was incubated overnight at 80° C. after adding 3N methanolic HCl (2 ml).

The sample was allowed to cool to room temperature, and 0.9% NaCl (1 ml)was added. The mixture was vortexed after adding n-hexane (2 ml). Thiswas followed by centrifugation at 3,000 rpm for 10 min, and the upperlayer was collected. The mixture was further vortexed after addingn-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upperlayer was collected. After adding a small amount of anhydrous sodiumsulfate to the collected upper layer, the mixture was vortexed, andcentrifuged at 3,000 rpm for 10 min. After collecting the upper layer,the upper layer was concentrated and dried with nitrogen gas. Theconcentrated dry sample was dissolved in n-hexane (0.5 ml), and 1 μl ofthe sample was GC analyzed. The GC analysis was performed with a gaschromatograph GC-2014 (Shimadzu Corporation) under the followingconditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

As a result, knocking out the C20 elongase in the Parietichytriumsarkarianum SEK364 caused reduction of fatty acids of 22 or greatercarbon chain length, and increased fatty acids of 20 carbon chain length(FIG. 21). FIG. 22 represents the proportions relative to the wild-typestrain taken as 100%. FIG. 22 represents the proportion of eachcomponent based on the total amount of the fatty acids, which includesAA: about 25.2%, DGLA: about 8.6%, ETA: about 0.6%, EPA: about 11.6%,n-6DPA: about 1.6%, and DHA: about 1.3%, which can also be described asthe values of GC area such as n-6DPA/DTAL: 2.4, DHA/n-3DPA: 4.9,C20PUFA/C22PUFA: 11.9, and n-6PUFA/n-3PUFA 2.6. As can be seen fromthese results, the arachidonic acid increased about ten-fold, and theEPA showed about an eight-fold increase. The DPA and DHA reduced toabout ¼ and about ⅕, respectively.

Example 4

[Disruption of Thraustochytrium aureum PUFA PKS Pathway-Associated GeneOrfA]

[Example 4-1] Cloning of PUFA PKS Pathway-Associated Gene OrfA UpstreamSequence

Genomic DNA was extracted from the Thraustochytrium aureum ATCC 34304 byusing the method described in Example 3-2, and the DNA concentration wascalculated by measuring A260/280. By using this, a genome cassettelibrary was produced with an LA PCR™ in vitro Cloning Kit (Takara Bio).A PCR lower primer [RHO20: 23mer: 5′-CGA TGA AAG GTC ACA GAA GAG TC-3′(SEQ ID NO: 89)] was set on the PUFA PKS pathway-associated gene OrfAdescribed in Patent Document 7, and the DNA was amplified by using thisprimer in combination with the cassette primer attached to the kit [1stPCR cycles: 98° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30cycles/72° C. 5 min]. The 1st PCR amplification product was diluted 100times, and amplified with the PCR lower primer [RHO20] and the nestedprimer attached to the kit [2nd PCR cycles: 98° C. 2 min/98° C. 30 sec,56° C. 30 sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The resulting DNAfragment was cloned into a pGEM-T easy vector, amplified withEscherichia coli, and the sequence was confirmed by using a DyeTerminator Cycle Sequencing Kit (BECKMAN COULTER).

The 3,377-bp (SEQ ID NO: 91) DNA fragment containing the upstream 3,181bp (SEQ ID NO: 90) of OrfA was cloned. The OrfA upstream DNA sequencewas found to be 3,181 bp.

[Example 4-2] Cloning of PUFA PKS Pathway-Associated Gene OrfADownstream Sequence

The genome cassette library produced in Example 4-1 was used as atemplate. The DNA was amplified by using the method described in Example4-1, using a PCR upper primer [RHO21: 21mer: 5′-CAG GGC GAG CGA GTG TGGTTC-3′ (SEQ ID NO: 92)] set on the PUFA PKS pathway-associated gene OrfAdescribed in Patent Document 7. The resulting DNA fragment was clonedinto a pGEM-T easy vector, amplified with Escherichia coli, and thesequence was confirmed by using a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER). The 1, 204-bp DNA fragment (SEQ ID NO: 94) containingthe downstream 1, 160 bp (SEQ ID NO: 93) of OrfA was cloned.

The DNA was amplified by using the method described in Example 4-1 usingthe PCR upper primer [RHO28: 20mer: 5′-TGA TGC CGA TGC TAC AAA AG-3′(SEQ ID NO: 95] produced on SEQ ID NO: 94. The resulting DNA fragmentwas cloned into a pGEM-T easy vector, amplified with Escherichia coli,and the sequence was confirmed by using a Dye Terminator CycleSequencing Kit (BECKMAN COULTER).

The 1,488-bp DNA fragment (SEQ ID NO: 96) containing the downstreamsequence was cloned. The downstream DNA sequence of OrfA was found to be2,551 bp in total (SEQ ID NO: 97).

[Example 4-3] Production of PUFA PKS Pathway-Associated Gene OrfATargeting Vector

By using the genomic DNA of Thraustochytrium aureum ATCC 34304 as atemplate, an 18S rDNA sequence (1,835 bp, SEQ ID NO: 98) was amplifiedwith a PrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers usedare as follows. TMO30 was set on the 18S rDNA sequence. TMO31 containsthe 18S rDNA sequence and an EF1α promoter sequence [TMO30: 30mer:5′-CGA ATA TTC CTG GTT GAT CCT GCC AGT AGT-3′ (SEQ ID NO: 99), TMO31:46mer: 5′-GTA ACG GCT TTT TTT GAA TTG CAG GTT CAC TAC GCT TGT TAG AAAC-3′ (SEQ ID NO: 100)] [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C.30 sec, 72° C. 2 min, 30 cycles/72° C. 2 min]. Separately, by using theThraustochytrium aureum ATCC 34304 genomic DNA as a template, the EF1αpromoter sequence (661 bp, SEQ ID NO: 101) was amplified with aPrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers used are asfollows. TMO32 contains the 18S rDNA sequence and the EF1α promotersequence. TMO33 contains the EF1α promoter sequence and an artificialneomycin-resistant gene sequence [TMO32: 46mer: 5′-GGT TTC CGT AGT GAACCT GCA ATT CAA AAA AAG CCG TTA CTC ACA T-3′ (SEQ ID NO: 102), TMO33:46mer: 5′-GCG TGA AGG CCG TCC TGT TCA ATC ATC TAG CCT TCC TTT GCC GCTG-3′ (SEQ ID NO: 103)] [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C.30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].

By using the artificial neomycin-resistant gene as a template, theartificial neomycin-resistant gene sequence (835 bp, SEQ ID NO: 104) wasamplified with a PrimeSTAR HS DNA polymerase (TakaraBio). The PCRprimers used are as follows. TMO34 contains the EF1α promoter sequenceand the artificial neomycin-resistant gene sequence. TMO35 contains theartificial neomycin-resistant gene sequence and the EF1α terminatorsequence [TMO34: 45mer: 5′-CAT CGG CAA AGG AAG GCT AGA TGA TTG AAC AGGACG GCC TTC ACG-3′ (SEQ ID NO: 105), TMO 35: 46mer: 5′-GCG CAT AGC CGGCGC GGA TCT CAA AAG AAC TCG TCC AGG AGG CGG T-3′ (SEQ ID NO: 106)] [PCRcycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 1 min, 30cycles/72° C. 1 min].

Further, by using the Thraustochytrium aureum ATCC 34304 genomic DNA asa template, the EF1α terminator sequence (1249 bp, SEQ ID NO: 107) wasamplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The PCRprimers used are as follows. TMO36 contains the artificialneomycin-resistant gene sequence and the EF1α terminator sequence. TMO37was set within the EF1α terminator [TMO36: 46mer: 5′-TCC TGG ACG AGT TCTTTT GAG ATC CGC GCC GGC TAT GCG CCC GTG C-3′ (SEQ ID NO: 108), TMO37:30mer: 5′-CAC TGC AGC GAA AGA CGG GCC GTA AGG ACG-3′ (SEQ ID NO: 109)][PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 2 min,30 cycles/72° C. 2 min].

By using SEQ ID NOS: 98, 101, 104, and 107 as templates, a fusion PCRwas performed according to the method described in Non-Patent Document19. An LA taq Hot start version (Takara Bio) was used as the enzyme. TheTMO30 (SEQ ID NO: 99) and TMO33 (SEQ ID NO: 103) set, and the TMO34 (SEQID NO: 105) and TMO37 (SEQ ID NO: 109) set were used for the firstamplification. The TMO30 (SEQ ID NO: 99) and TMO37 (SEQ ID NO: 109) setwas used for the second amplification. The PCR reaction was performed ata denature temperature of 98° C. for 10 seconds, and the annealing andthe extension reaction were appropriately adjusted according to theprimer Tm value and the amplification fragment length (FIG. 23).

The DNA fragment (FIG. 23, SEQ ID NO: 110, 4,453 bp) joined as above wascut at the EcoRI site of the T. aureum 18S rDNA, and the NcoI site ofthe T. aureum EF1α terminator, and ligated to a pGEM-T easyvector-derived vector. This was named pRH5 (FIG. 24).

By using the Thraustochytrium aureum ATCC 34304 genomic DNA as atemplate, the DNA was amplified with a PrimeSTAR HS DNA polymerase withGC Buffer (Takara Bio), using PCR primers set in the upstream sequencefound in Example 4-1 (SEQ ID NO: 90, and PUFA PKS pathway-associatedgene OrfA described in Patent Document 7). The amplification yielded a1,218-bp DNA fragment (SEQ ID NO: 111). This was used as the 5′homologous region of the targeting vector. The PCR primers used are asfollows. An EcoRI site or a HindIII site was added as a linker sequence[RHO33: 32mer: 5′-CCC GAA TTC GGA CGA TGA CTG ACT GAC TGA TT-3′ (SEQ IDNO: 112), RHO34: 28mer: 5′-CCC AAG CTT GTC TGC CTC GGC TCT TGG T-3′ (SEQID NO: 113)] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 57° C. 30 sec, 72°C. 1 min, 30 cycles/72° C. 3 min].

By using the Thraustochytrium aureum ATCC 34304 genomic DNA as atemplate, the DNA was amplified with a PrimeSTAR HS DNA polymerase withGC Buffer (Takara Bio) using the PCR primers set in the downstreamsequence (SEQ ID NO: 97) found in Example 4-2. The amplification yieldeda 1,000-bp DNA fragment (SEQ ID NO: 114). This was used as the 3′homologous region of the targeting vector. The PCR primers used are asfollows. A linker sequence NcoI site was added to the both primers[RHO29: 28mer: 5′-CCC CCA TGG TGT TGC TGT GGG ATT GGT C-3′ (SEQ ID NO:115), RHO30: 30mer: 5′-CCC CCA TGG CTC GGT TAC ATC TCT GAG GAA-3′ (SEQID NO: 116)] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 57° C. 30 sec, 72°C. 1 min, 30 cycles/72° C. 3 min].

The amplified upstream sequence was joined to the EcoRI site and theHindIII site in the pRH5 of FIG. 24. The amplified downstream sequencewas joined to the NcoI site. This vector was named pRH21.

The produced targeting vector (pRH21) using the artificialneomycin-resistant gene is shown in FIG. 25.

[Example 4-4] Production of PUFA PKS Pathway-Associated Gene OrfATargeting Vector (Hygromycin-Resistant Gene)

By using the pRH32 (FIG. 15) of Example 3-3 as a template, a ubiqitinpromoter-hygromycin-resistant gene fragment (1,632 bp, SEQ ID NO: 117)was amplified with a PrimeSTAR HS DNA polymerase with GC Buffer (TakaraBio). The PCR primers used are as follows. RHO59 was set on theubiquitin promoter, and a linker sequence HindIII site was added. RHO60contains a hygromycin-resistant gene sequence stop codon, and has linkersequences SphI and SalI [RHO59: 36mer: 5′-CCC AAG CTT GCC GCA GCG CCTGGT GCA CCC GCC GGG-3′ (SEQ ID NO: 118), RHO60: 43mer: 5′-CCC GCA TGCGTC GAC TAT TCC TTT GCC CTC GGA CGA GTG CTG G-3′ (SEQ ID NO: 119)] [PCRcycles: 98° C. 2 min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2min].

The amplified fragment was joined to the HindIII and SphI sites of thepRH21 (FIG. 25) of Example 4-3 (FIG. 26, pRH30).

By using the Thraustochytrium aureum ATCC 34304 genomic DNA as atemplate, the gene was amplified with a PrimeSTAR HS DNA polymerase withGC Buffer (Takara Bio) using the PCR primers produced in the downstreamsequence (SEQ ID NO: 97) found in Example 4-2. The amplification yieldeda 1,000-bp DNA fragment (SEQ ID NO: 120). This was used as the 3′homologous region of the targeting vector. The PCR primers used are asfollows. A linker sequence SalI site was added to the both primers[RHO61: 29mer: 5′-CCC GTC GAC GTG TTG CTG TGG GAT TGG TC-3′ (SEQ ID NO:121), RHO62: 29mer: 5′-CCC GTC GAC TCG GTT ACA TCT CTG AGG AA-3′ (SEQ IDNO: 122)] [PCR cycles: 98° C. 2 min/98° C. 30 sec, 57° C. 30 sec, 72° C.1 min, 30 cycles/72° C. 3 min].

The amplified downstream sequence was joined to the SalI site of pRH30(FIG. 26). This was named pRH33. The produced targeting vector (pRH33)using the hygromycin-resistant gene is shown in FIG. 27.

[Example 4-5] Introduction of PUFA PKS Pathway-Associated Gene OrfATargeting Vector

By using the targeting vectors produced in Examples 4-3 and 4-4 astemplates, the gene was amplified with a PrimeSTAR Max DNA polymerase(Takara Bio) using the RHO30 (Example 4-3; SEQ ID NO: 116) and RHO33(Example 4-3; SEQ ID NO: 112) as primers [PCR cycles: 98° C. 2 min/98°C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min]. Afterbeing extracted with phenol-chloroform and then with chloroform, the DNAwas precipitated with ethanol, and the precipitate was dissolved in0.1×TE. The DNA concentration was calculated by measuring A260/280. Theintroduced fragment obtained from using the pRH21 (FIG. 25) of Example4-3 as a template was 3,705 bp, and had the following sequence order:Thraustochytrium aureum OrfA gene upstream-EF1α promotersequence-artificial neomycin-resistant gene sequence-Thraustochytriumaureum OrfA gene downstream (SEQ ID NO: 123). The introduced fragmentobtained from using the pRH33 (FIG. 27) of Example 4-4 as a template was3,826 bp, and had the following sequence order: Upstream ofThraustochytrium aureum OrfA gene-ubiquitin promotersequence-hygromycin-resistant gene sequence-downstream ofThraustochytrium aureum OrfA gene (SEQ ID NO: 124).

The Thraustochytrium aureum ATCC 34304 strain was cultured in a GYmedium for 4 days, and cells in the logarithmic growth phase were usedfor gene introduction. The DNA fragment (0.625 μg) was then introducedinto cells corresponding to OD600=1 to 1.5 using the gene-gun technique(microcarrier: 0.6-micron gold particles, target distance: 6 cm, chambervacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 4- to 6-hour recoverytime, the cells with the introduced gene were applied onto a PDA agarplate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin). As aresult, 100 to 200 drug resistant strains were obtained per penetration.

[Example 4-6] Identification of PUFA PKS Pathway-Associated Gene OrfAGene Targeting Homologous Recombinant

Genomic DNA was extracted from the Thraustochytrium aureum ATCC 34304,the hetero homologous recombinant, and the homo homologous recombinant(PKS pathway-associated gene disrupted strain) by using the methoddescribed in Example 3-2. The DNA concentration was calculated bymeasuring A260/280.

The genomic DNA was cut with restriction enzymes, and electrophoresed inabout 2 to 3 μg per well with a 0.7% SeaKem GTG agarose gel (TakaraBio). This was transferred to a nylon membrane, and hybridized at 54° C.for 16 hours with the probes produced by using a DIG system (RocheApplied Science). The following primers were used for the probeproduction.

5′ end (SEQ ID NO: 125) [RHO37: 22mer: 5′-GAA GCG TCC CGT AGA TGT GGTC-3′, (SEQ ID NO: 126) RH038: 21mer: 5′-GCC CGA GAG GTC AAA GTA CGC-3′]3′ end (SEQ ID NO: 127) [RHO39: 20mer: 5′-GCG AGC CCA GGT CCA CTT GC-3′,(SEQ ID NO: 128) RHO40: 22mer: 5′-CAG CCC GAT GAA AAA CTT GGT  C-3′]PCR cycles: 98° C. 2 min/98° C. 30 sec,60° C. 30 sec, 72° C. 2 min, 30 cycles/ 72° C. 3 min

The restriction enzymes used and the probe positions are as shown inFIG. 28. Detection of the hybridized probes was made by using thechromogenic method (NBT/BCIP solution).

Bands of the sizes expected from the homologous recombination of thedrug resistant genes were observed in the analyses of both the 5′ endand the 3′ end (FIG. 29).

[Example 4-7] Changes in Fatty Acid Composition by Disruption of PUFAPKS Pathway-Associated Gene OrfA

The Thraustochytrium aureum ATCC 34304 and the gene disrupted strainwere cultured and freeze dried according to the methods of Example 3-9,and the fatty acids were methylesterificated, and GC analyzed.

FIG. 30 represents changes in fatty acid composition. FIG. 31 representsthe proportions relative to the wild-type strain taken as 100%. FIG. 31represents the proportion of each component based on the total amount ofthe fatty acids, which includes AA: 3.1%, DGLA: 0.2%, ETA: 0.04%, EPA:6.8%, n-6DPA: 10.7%, and DHA: 22.6%, which can also be described as thevalues of GC area such as n-6DPA/DTA: 5.3, DHA/n-3DPA: 20.0,C20PUFA/C22PUFA: 0.3, and n-6PUFA/n-3PUFA: 0.5. As can be seen fromthese results, disrupting the PUFA PKS pathway-associated gene OrfA inthe Thraustochytrium aureum tended to increase the DPA (C22: 5n-6) anddecrease the DHA (C22: 6n-3).

Example 5

[Disruption of C20 Elongase Gene in Thraustochytrium aureum OrfADisrupted Strain]

[Example 5-1] Cloning of Upstream Sequence of Thraustochytrium aureumC20 Elongase Gene

The genome cassette library produced in Example 4-1 was used as atemplate. A PCR lower primer [RHO71: 22mer: 5′-GGG AGC GCA GGG AAA ACGGTC T-3′ (SEQ ID NO: 129)] was produced on the C20 elongase geneupstream sequence (SEQ ID NO: 24) of Example 2-5, and the gene wasamplified by using this primer with the cassette primer attached to thekit used in Example 4-1 [1st PCR cycles: 98° C. 2 min/98° C. 30 sec, 56°C. 30 sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The 1st PCRamplification product was diluted 100 times, and the gene was amplifiedby using the PCR lower primer [RHO72: 20mer: 5′-CCA GCC CAC GTC GTC GGAGC-3′ (SEQ ID NO: 130)] with the nested primer attached to the kit usedin Example 4-1 [2nd PCR cycles: 98° C. 2 min/98° C. 30 sec, 56° C. 30sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The resulting DNA fragmentwas cloned into a pGEM-T easy vector, amplified with Escherichia coli,and the sequence was confirmed by using a Dye Terminator CycleSequencing Kit (BECKMAN COULTER).

The 2,297-bp DNA fragment (SEQ ID NO: 131) containing the upstream-3,277bp to −981 bp region of the C20 elongase gene was cloned.

[Example 5-2] Cloning of C20 Elongase Gene Downstream Sequence

The genome cassette library produced in Example 4-1 was used as atemplate. A PCR upper primer [RHO87: 23 mer: 5′-GCC GCT CAT GCC CAC GCTCAA AC-3′ (SEQ ID NO: 132)] was produced on the C20 elongase genedownstream sequence (SEQ ID NO: 25) of Example 2-5, and the gene wasamplified by using this primer with the cassette primer attached to thekit used in Example 4-1 [1st PCR cycles: 98° C. 2 min/98° C. 30 sec, 56°C. 30 sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The 1st PCRamplification product was diluted 100 times, and the gene was amplifiedby using the PCR lower primer [RHO73: 23 mer: 5′-CTT TCG GCT GCC AGG AATCTA CG-3′ (SEQ ID NO: 133)] with the nested primer attached to the kitused in Example 4-1 [2nd PCR cycles: 98° C. 2 min/98° C. 30 sec, 56° C.30 sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The resulting DNAfragment was cloned into a pGEM-T easy vector, amplified withEscherichia coli, and the sequence was confirmed by using a DyeTerminator Cycle Sequencing Kit (BECKMAN COULTER).

The 2,189-bp DNA fragment (SEQ ID NO: 134) containing the downstream1,106 bp to 3,294 bp region of the C20 elongase gene was cloned.

[Example 5-3] Production of Blasticidin-Resistant Gene Cassette

A ubiquitin promoter sequence (618 bp, SEQ ID NO:135) was amplified witha PrimeSTAR HS DNA polymerase with GC Buffer (Takara Bio), using theThraustochytrium aureum ATCC 34304 genomic DNA as a template. The PCRprimers used are as follows. RHO53 was set on the ubiquitin promotersequence, and contains a BglII linker sequence (Example 3-2, SEQ ID NO:55). RHO48 contains the ubiquitin promoter sequence and ablasticidin-resistant gene sequence [RHO48: 58mer: 5′-CTT CTT GAG ACAAAG GCT TGG CCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3′ (SEQ IDNO: 136)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30cycles/68° C. 1 min].

By using pTracer-CMV/Bsd/lacZ as a template, the blasticidin-resistantgene (432 bp, SEQ ID NO: 137) was amplified with a PrimeSTAR HS DNApolymerase with GC Buffer. The PCR primers used are as follows. RHO47contains the ubiquitin promoter sequence and the blasticidin-resistantgene sequence. RHO49 contains the blasticidin-resistant gene sequence,and has a BglII linker sequence [RHO47: 54mer:5′-AGC GAC CTA AGC AAC ACTAGC CAA CAT GGC CAA GCC TTT GTC TCA AGA AGA ATC-3′ (SEQ ID NO: 138),RHO49: 38mer: 5′-CCC AGA TCT TAG CCC TCC CAC ACA TAA CCA GAG GGC AG-3′(SEQ ID NO: 139)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min,30 cycles/68° C. 1 min].

By using SEQ ID NOS: 135 and 137 as templates, a fusion PCR wasperformed with RHO53 (Example 3-2, SEQ ID NO: 55) and RHO49 (SEQ ID NO:139) according to the method described in Non-Patent Document 19. An LAtaq Hot start version (Takara Bio) was used as the enzyme. After theamplification performed under the following conditions, the product wasdigested with BglII [PCR cycles: 94° C. 2 min/94° C. 20 sec, 55° C. 30sec, 68° C. 1 min, 30 cycles/68° C. 1 min (1° C./10 sec from 55° C. to68° C.)] (FIG. 32).

The fused Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter-pTracer-CMV/Bsd/lacZ-derived blasticidin-resistant gene (1,000bp, SEQ ID NO: 140) was digested with BglII, and ligated to the BamHIsite of the pRH27 (FIG. 11) of Example 3-1. The resulting plasmid wasamplified with Escherichia coli, and the sequence was confirmed by usinga Dye Terminator Cycle Sequencing Kit (BECKMAN COULTER). This was namedpRH38.

The product blasticidin-resistant gene cassette (pRH38) is shown in FIG.33.

[Example 5-4] Production of GFP-Fused Zeocin-Resistant Gene Cassette

By using the Thraustochytrium aureum ATCC 34304 genomic DNA as atemplate, a ubiquitin promoter sequence (812 bp, SEQ ID NO: 141) wasamplified with a PrimeSTAR HS DNA polymerase with GC Buffer (TakaraBio). The PCR primers used are as follows. TMO38 was set on theubiquitin promoter sequence. TMO39 contains the ubiquitin promotersequence and an enhanced GFP gene sequence [TMO38: 29mer: 5′-TCG GTA CCCGTT AGA ACG CGT AAT ACG AC-3′ (SEQ ID NO: 142), TMO39: 41mer: 5′-TCC TCGCCC TTG CTC ACC ATG TTG GCT AGT GTT GCT TAG GT-3′ (SEQ ID NO: 143)] [PCRcycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 1 min, 30cycles/72° C. 1 min].

By using the enhanced GFP gene sequence (clontech) as a template, anenhanced GFP gene sequence (748 bp, SEQ ID NO: 144) was amplified with aPrimeSTAR HS DNA polymerase (Takara Bio). The PCR primers used are asfollows. TMO40 contains the ubiquitin promoter sequence and the enhancedGFP gene sequence. RHO91 contains the enhanced GFP sequence and azeocin-resistant gene sequence [TMO40: 41mer: 5′-ACC TAA GCA ACA CTA GCCAAC ATG GTG AGC AAG GGC GAG GA-3′ (SEQ ID NO: 145), RHO91: 58mer: 5′-GAACGG CAC TGG TCA ACT TGG CGT CCA TGC CGA GAG TGA TCC CGG CGG CGG TCA CGAA-3′ (SEQ ID NO: 146)] [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C.30 sec, 72° C. 2 min, 30 cycles/72° C. 2 min].

By using SEQ ID NOS: 141 and 144 as templates, a fusion PCR wasperformed with an LA taq Hot start version (Takara Bio) according to themethod described in Non-Patent Document 19. TMO38 (SEQ ID NO: 142) andRHO91 (SEQ ID NO: 146) were used as primers, and the reaction wasperformed under the following conditions [PCR cycles: 94° C. 2 min/94°C. 20 sec, 55° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min (1° C./10sec from 55° C. to 68° C.)] (FIG. 34, 1,519 bp, SEQ ID NO: 147).

By using SEQ ID NO: 147 as a template, the ubiquitin promotersequence-enhanced GFP gene sequence (1,319 bp, SEQ ID NO: 148) wasamplified with a PrimeSTAR HS DNA polymerase (Takara Bio). The primersused are as follows. RHO53 (Example 3-2, SEQ ID NO: 55) contains theubiquitin promoter sequence, and has a BglII site. RHO91 (SEQ ID NO:146) contains the enhanced GFP sequence and the zeocin-resistant genesequence [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 2 min, 30cycles/68° C. 2 min].

By using pcDNA3.1 Zeo(+) as a template, the zeocin-resistant genesequence (408 bp, SEQ ID NO: 149) was amplified with a PrimeSTAR HS DNApolymerase (Takara Bio). RHO92 contains the enhanced GFP sequence andthe zeocin-resistant gene sequence. RHO64 contains the zeocin-resistantgene sequence, and has a BglII site [RHO92: 54mer: 5′-CGC CGC CGG GATCAC TCT CGG CAT GGA CGC CAA GTT GAC CAG TGC CGT TCC GGT-3′ (SEQ ID NO:150), RHO64: 38mer: 5′-CCC AGA TCT CAG TCC TGC TCC TCG GCC ACG AAG TGCAC-3′ (SEQ ID NO: 151)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C.1 min, 30 cycles/68° C. 1 min].

By using SEQ ID NOS: 148 and 149 as templates, a fusion PCR wasperformed with an LA taq Hot start version (Takara Bio) according to themethod described in Non-Patent Document 19. RHO53 (Example 3-2, SEQ IDNO: 55) and RHO64 (SEQ ID NO: 151) were used as primers, and thereaction was performed under the following conditions [PCR cycles: 94°C. 2 min/94° C. 20 sec, 68° C. 2 min, 30 cycles/68° C. 2 min (1° C./10sec from 55° C. to 68° C.)] (FIG. 35).

The fused Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter-enhanced GFP gene-pcDNA3.1 Zeo(+)-derived zeocin-resistant gene(FIG. 35, 1,677 bp, SEQ ID NO: 152) was digested with BglII, and ligatedto the BamHI site of the pRH27 (FIG. 11) of Example 3-1. The resultingplasmid was amplified with Escherichia coli, and the sequence wasconfirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMANCOULTER). This was named pRH51.

The product GFP-fused zeocin-resistant gene cassette (pRH51) is shown inFIG. 36.

[Example 5-5] Production of Base Plasmid for C20 Elongase Gene TargetingVector Production

By using the Thraustochytrium aureum ATCC 34304 genomic DNA as atemplate, the C20 elongase gene and the nearby sequences (2,884 bp, SEQID NO: 153) were PCR amplified with a PrimeSTAR HS DNA polymerase(Takara Bio). The PCR primers used are as follows. The both primerscontain EcoRI linker sequences. KSO9 was set upstream of the C20elongase gene (SEQ ID NO: 24), and KSO10 downstream of the C20 elongasegene (SEQ ID NO: 25) [KSO9: 50mer: 5′-CCC GAA TTC ACT AGT GAT TCT CCCGGG TGG ACC TAG CGC GTG TGT CAC CT-3′ (SEQ ID NO: 154), KSO10: 40mer:5′-CCC GAA TTC GAT TAT CCC GGG GCC GAG AAC GGG GTC GCC C-3′ (SEQ ID NO:155)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3.5 min, 30cycles/68° C. 10 min]. A PrimeSTAR HS DNA Polymerase (Takara Bio) wasused as the enzyme. After the amplification, the product was digestedwith EcoRI, and cloned into the EcoRI site of the pBluescript(SK)(stratagene) vector. After amplification with Escherichia coli, thesequence was confirmed by using a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER) (FIG. 37).

By using the plasmid of FIG. 37 as a template, amplification wasperformed with a PrimeSTAR Max DNA Polymerase (TakaraBio), using aprimer set of the reverse orientation prepared for the deletion of theC20 elongase gene sequence portion and the insertion of a BglII site(1,939 bp, SEQ ID NO: 156). The PCR primers used are as follows. Theboth primers have BglII linker sequences [RHO69: 38mer: 5′-CCC AGA TCTACC TGT TTC CGG CTG GCT CCC GAG CCA TG-3′ (SEQ ID NO: 157), RHO70:38mer: 5′-CCC AGA TCT GGT CGC GTT TAC AAA GCA GCG CAG CAA CA-3′ (SEQ IDNO: 158)] [PCR cycles: 98° C. 2 min/98° C. sec, 68° C. 1.5 min, 30cycles/68° C. 1.5 min]. After the amplification performed under theseconditions, the product was digested with BglII, and allowed to selfligate. The ligated sample was amplified with Escherichia coli, and thesequence was confirmed with a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER). This was named pRH40.

The produced base plasmid (pRH40) for the production of the C20 elongasegene targeting vector is shown in FIG. 38.

[Example 5-6] Production of Targeting Vectors (Blasticidin-ResistantGene and GFP-Fused Zeocin-Resistant Gene)

The pRH38 (FIG. 33) of Example 5-3 was digested with BglII, and the DNAfragment containing the blasticidin-resistant gene cassette was ligatedto the BglII site of the pRH40 (FIG. 38) of Example 5-5. This was namedpRH43.

The pRH51 (FIG. 36) of Example 5-4 was digested with BglII, and the DNAfragment containing the GFP-fused zeocin-resistant gene cassette wasligated to the BglII site of the pRH40 (FIG. 38) of Example 5-5. Thiswas named pRH54.

The two targeting vectors (pRH43 and 54) produced are shown in FIG. 39.

[Example 5-7] Introduction of C20 Elongase Gene Targeting Vector intoThraustochytrium aureum OrfA Disrupted Strain

By using the two targeting vectors produced in Example 5-6 as templates,the gene was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio),using KSO11 and KSO12 as primers. KSO11 was set upstream of theThraustochytrium aureum C20 elongase gene, and KSO12 downstream of theThraustochytrium aureum C20 elongase gene [KSO11: 31mer: 5′-CTC CCG GGTGGA CCT AGC GCG TGT GTC ACC T-3′ (SEQ ID NO: 159), KSO12: 27mer: 5′-ATCCCG GGG CCG AGA ACG CCC TCG CCC-3′ (SEQ ID NO: 160)] [PCR cycles: 98° C.2 min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min]. After beingextracted with phenol-chloroform and then with chloroform, the DNA wasprecipitated with ethanol, and the precipitate was dissolved in 0.1×TE.The DNA concentration was calculated by measuring A260/280. Theintroduced fragment obtained from using the pRH43 (FIG. 39) of Example5-6 as a template was 3,215 bp, and had the following sequence order:Upstream of Thraustochytrium aureum C20 elongase gene-ubiquitinpromoter-blasticidin-resistant gene sequence-SV40 terminatorsequence-downstream of Thraustochytrium aureum C20 elongase gene (SEQ IDNO: 161). The introduced fragment obtained from using the pRH54 (FIG.39) of Example 5-6 as a template was 3,887 bp, and had the followingsequence order: Upstream of Thraustochytrium aureum C20 elongasegene-ubiquitin promoter-enhanced GFP gene sequence-zeocin-resistant genesequence-SV40 terminator sequence-downstream of Thraustochytrium aureumC20 elongase gene (SEQ ID NO: 162).

The disrupted strain of the PUFA PKS pathway-associated gene OrfA genedescribed in Example 4 was cultured in a GY medium for 4 days, and cellsin the logarithmic growth phase were used for gene introduction. The DNAfragment (0.625 μg) was then introduced into cells corresponding toOD600=1 to 1.5 using the gene-gun technique (microcarrier: 0.6-microngold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupturedisk: 1,100 PSI). After a 4- to 6-hour recovery time, the cells with theintroduced gene were applied onto a PDA agar plate medium (containing 2mg/ml G418 or 2 mg/ml hygromycin). As a result, 100 to 200 drugresistant strains were obtained per penetration.

[Example 5-8] Identification of C20 Elongase Gene Gene TargetingHomologous Recombinant

Genomic DNA was extracted from the Thraustochytrium aureum and the C20elongase gene disrupted strain of the Thraustochytrium aureum OrfAdisrupted strain by using the method described in Example 3-2. The DNAconcentration was calculated by measuring A260/280.

The genomic DNA was cut with restriction enzymes, and electrophoresed inabout 2 to 3 μg per well in a 0.7% SeaKem GTG agarose gel (Takara Bio).This was transferred to a nylon membrane, and hybridized at 51° C. for16 hours with the probes produced by using a DIG system (Roche AppliedScience). The following primers were used for the probe production.

5′ end [RHO94: 21mer: (SEQ ID NO: 163)5′-ACG TCC GCT TCA AAC ACC TCG-3′, RHO95: 24mer: (SEQ ID NO: 164)5′-TCG GAA CAA CTG GAA CAA CTA AAG-3′] 3′ end [RHO96: 22mer:(SEQ ID NO: 165) 5′-ATG TCG CTC TCC TTC TTC TCA G-3′, RHO97: 21mer:(SEQ ID NO: 166) 5′-TCG GCT CCT GGA AAG TGC TCT-3′] PCR cycles:98° C. 2 min/98° C. 30 sec, 58° C. 30 sec, 72° C. 1 min,30 cycles/72° C. 3 min

The restriction enzymes used and the probe positions are as shown inFIG. 40. Detection of the hybridized probes was made by using achromogenic method (NBT/BCIP solution).

Bands of the sizes expected from the homologous recombination of thedrug resistant genes were observed in the analyses of both the 5′ endand the 3′ end (FIG. 41). It was found by the experiment that theThraustochytrium aureum ATCC 34304 strain did not become auxotrophiceven with the deletion of the PKS pathway-associated gene OrfA and theC20 elongase gene.

[Example 5-9] Changes in Fatty Acid Composition by Disruption of C20Elongase Gene in Thraustochytrium aureum OrfA Disrupted Strain

The Thraustochytrium aureum ATCC 34304 and the gene disrupted strainwere cultured and freeze dried according to the method of Example 3-9,and the fatty acids were methylesterificated, and GC analyzed. The GCanalysis was performed with a gas chromatograph GC-2014 (ShimadzuCorporation) under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

Changes in fatty acid composition are represented in FIG. 42. FIG. 43represents the proportions relative to the wild-type strain taken as100%. FIG. 43 represents the proportion of each component based on thetotal amount of the fatty acids, which includes AA: 19.5%, DGLA: 1.8%,ETA: 0.3%, EPA: 24.9%, n-6D PA: 5.9%, and DHA: 6.8%, which can also bedescribed as the values of GC area such as n-6DPA/DTA: 12.6, DHA/n-3DPA:11.7, C20PUFA/C22PUFA: 3.4, and n-6PUFA/n-3PUFA: 0.8.

As can be seen from these results, disrupting the C20 elongase gene inthe Thraustochytrium aureum OrfA disrupted strain increased the C20:4n-6(AA) about eight-fold, and the C20:5n3 (EPA) about four-fold, anddecreased the C22:6n-3 (DHA) to about ⅕.

Example 6

[Expression of ω3 Desaturase Gene in Thraustochytrium aureum OrfADisrupted Strain]

[Example 6-1] Cloning of Saprolegnia diclina-Derived ω3 Desaturase Geneand Production of Gene Expression Plasmid

Genomic DNA was extracted from the Thraustochytrium aureum ATCC 34304 byusing the method of Example 3-2, and the DNA concentration wascalculated by measuring A260/280. By using this as a template, theubiquitin promoter sequence (longer) (812 bp, SEQ ID NO: 167) wasamplified with an LA Taq with GC Buffer (Takara Bio, Buffer II wasused). The PCR primers used are as follows. TMO42 was set on theubiquitin promoter sequence, upstream of RHO53 (Example 3-2, SEQ ID NO:55), and contains a KpnI linker sequence. TMO43 contains the ubiquitinpromoter sequence and a Saprolegnia diclina-derived ω3 desaturase genesequence [TMO42: 29mer: 5′-TCG GTA CCC GTT AGA ACG CGT AAT ACG AC-3′(SEQ ID NO: 168), TMO43: 45mer: 5′-TTC GTC TTA TCC TCA GTC ATG TTG GCTAGT GTT GCT TAG GTC GCT-3′ (SEQ ID NO: 169)] [PCR cycles: 96° C. 2min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].

Then, Saprolegnia diclina was cultured in a medium (adjusted withdeionized water) containing D-Glucose (31.8 g) and yeast extract (10.6g) per liter. Cells in the late stage of the logarithmic growth phasewere centrifuged at 4° C., 3,500×g for 5 min to form a pellet, anddisrupted by being frozen with liquid nitrogen. After being extractedwith phenol, the cell disruption liquid was precipitated with ethanol,and the precipitate was dissolved in a TE solution. The nucleic acidsdissolved in the TE solution were treated with RNase at 37° C. for 30min to degrade RNA. After being reextracted with phenol, the product wasprecipitated with ethanol, and the precipitate was dissolved in a TEsolution. The DNA purity and concentration were calculated by measuringA260/280. By using the resulting Saprolegnia diclina genomic DNA as atemplate, the Saprolegnia diclina-derived ω3 desaturase gene sequence(1,116 bp, SEQ ID NO: 170) was amplified with an LA Taq with GC Buffer(Takara Bio, Buffer II was used). The PCR primers used are as follows.TMO44 contains the ubiquitin promoter sequence and the Saprolegniadiclina-derived ω3 desaturase gene sequence. TMO45 contains theSaprolegnia diclina-derived ω3 desaturase gene sequence and theubiquitin terminator [TMO44: 43mer: 5′-CCT AAG CAA CAC TAG CCA ACA TGACTG AGG ATA AGA CGA AGG T-3′ (SEQ ID NO: 171), TMO45: 40mer: 5′-ATA CTACAG ATA GCT TAG TTT TAG TCC GAC TTG GCC TTG G-3′ (SEQ ID NO: 172)] [PCRcycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 1 min 30 sec,30 cycles/72° C. 1 min 30 sec].

By using the Thraustochytrium aureum ATCC 34304 genomic DNA as atemplate, the ubiquitin terminator sequence (614 bp, SEQ ID NO: 173) wasamplified with an LA Taq with GC Buffer (Takara Bio, Buffer II wasused). The primers used are as follows. TMO46 contains the Saprolegniadiclina-derived ω3 desaturase gene sequence and the ubiquitinterminator. TMO47 was designed on the ubiquitin terminator sequence, andcontains a KpnI linker sequence [TMO46: 44mer: 5′-CCA AGG CCA AGT CGGACT AAA ACT AAG CTA TCT GTA GTA TGT GC-3′ (SEQ ID NO: 174), TMO47:45mer: 5′-TCG GTA CCA CCG CGT AAT ACG ACT CAC TAT AGG GAG ACT GCA GTT-3′(SEQ ID NO: 175)] [PCR cycles: 96° C. 2 min/98° C. 20 sec, 60° C. 30sec, 72° C. 1 min, 30 cycles/72° C. 1 min].

By using SEQ ID NOS: 167, 170, and 173 as templates, a fusion PCR wasperformed with TMO42 (SEQ ID NO: 168) and TMO47 (SEQ ID NO: 175)according to the method described in Non-Patent Document 19. An LA Taqwith GC Buffer (Takara Bio, Buffer II was used) was used as the enzyme,and the amplification was performed under the following conditions [PCRcycles: 96° C. 2 min/98° C. 20 sec, 55° C. 30 sec, 68° C. 3 min, 30cycles/68° C. 3 min (1° C./10 sec from 55° C. to 68° C.] (FIG. 44, 2,463bp, SEQ ID NO: 176).

By using the pRH38 (FIG. 33) of Example 5-3 as a template, a PCR wasperformed with RHO84 (SEQ ID NO: 177, the sequence is presented below)and RHO52 (Example 3-1, SEQ ID NO: 52). RHO84 was set on the ubiquitinpromoter, and has a KpnI linker sequence. RHO52 was set on the SV40terminator sequence, and has a BglII linker. An LA taq Hot start versionwas used as the enzyme, and, after the amplification performed under thefollowing conditions, the product was cloned into a pGEM-T easy vector[RHO84: 36mer: 5′-CCC GGT ACC GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′(SEQ ID NO: 177)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min30 sec, 30 cycles/68° C. 3 min]. After amplification with Escherichiacoli, the sequence was confirmed by using a Dye Terminator CycleSequencing Kit (BECKMAN COULTER). This was named pRH45 (FIG. 45).

The fused Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter-Saprolegnia diclina-derived ω3 desaturase gene-Thraustochytriumaureum ATCC 34304-derived ubiquitin terminator (SEQ ID NO: 176; FIG. 44)was digested with KpnI, and ligated to the KpnI site of pRH45 (FIG. 45).The resulting plasmid was amplified with Escherichia coli, and thesequence was confirmed by using a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER). This was named pRH48.

The product Saprolegnia diclina-derived ω3 desaturase gene expressionplasmid (pRH48) is shown in FIG. 46.

[Example 6-2] Introduction of Saprolegnia diclina-Derived ω3 DesaturaseExpression Plasmid into Thraustochytrium aureum OrfA Disrupted Strain

By using the targeting vector produced in Example 6-1 as a template, DNAwas amplified with a PrimeSTAR Max DNA polymerase (Takara Bio), usingTMO42 (SEQ ID NO: 168) and RHO52 (Example 3-1, SEQ ID NO: 52) as primers[PCR cycles: 94° C. 30 sec, 72° C. 1 min, 5 cycles/94° C. 30 sec, 70° C.30 sec, 72° C. 1 min, 5 cycles/94° C. 30 sec, 68° C. 30 sec, 72° C. 1min, 25 cycles/72° C. 2 min]. The amplification product was collectedfrom the 1.0% agarose gel, and precipitated with ethanol. Theprecipitate was then dissolved in 0.1×TE. The DNA concentration wascalculated by measuring A260/280. The introduced fragment obtained byPCR was 3,777 bp, and had the following sequence order: ubiquitinpromoter-ω3 desaturase gene-ubiquitin terminator-ubiquitinpromoter-blasticidin-resistant gene sequence-SV40 terminator sequence(SEQ ID NO: 178).

The Thraustochytrium aureum OrfA disrupted strain produced in Example 4was cultured in a GY medium for 4 days, and cells in the logarithmicgrowth phase were used for gene introduction. The DNA fragment (0.625μg) was then introduced into cells corresponding to OD600=1 to 1.5 usingthe gene-gun technique (microcarrier: 0.6-micron gold particles, targetdistance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 1,100 PSI). Aftera 4- to 6-hour recovery time, the cells with the introduced gene wereapplied to a PDA agar plate medium (containing 0.2 mg/ml blasticidin).As a result, 20 to 30 drug resistant strains were obtained perpenetration.

[Example 6-3] Acquisition of Saprolegnia diclina-Derived ω3 DesaturaseGene Expression Strain

Genomic DNA was extracted from the Thraustochytrium aureum OrfAdisrupted strain produced in Example 3 and the ω3 desaturase geneexpressing strain by using the method described in Example 3-2. The DNAconcentration was calculated by measuring A260/280. By using this as atemplate, a PCR was performed with an LA taq Hot start version toconfirm the genome structure. The positions of the primers, combinationsused for the amplification, and the expected size of the amplificationproduct are shown in FIG. 47. TMO42 (Example 6-1, SEQ ID NO: 168) wasset on the ubiquitin promoter. RHO49 (Example 5-3, SEQ ID NO: 139) wasset on the blasticidin-resistant gene [PCR cycles: 98° C. 2 min/98° C.10 sec, 68° C. 4 min, 30 cycles/68° C. 7 min].

The result of the amplification confirmed a band of the expected size(FIG. 48). That is, a strain was isolated that contained the introducedexpression fragment stably introduced into its genome.

[Example 6-4] Changes in Fatty Acid Composition by ω3 DesaturaseExpression in PUFA PKS Pathway Disrupted Strain

The Thraustochytrium aureum OrfA disrupted strain produced in Example 4,and the ω3 desaturase expressing strain produced in Example 6-3 werecultured by using the method described in Example 3-9. After freezedrying, the fatty acids were methylesterificated, and GC analyzed. TheGC analysis was performed with a gas chromatograph GC-2014 (ShimadzuCorporation) under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

The ω3 desaturase expressing strain had reduced levels of the n-6 seriesfatty acids, and there was a tendency for the n-3 series fatty acids toincrease (FIG. 49). FIG. 50 represents the proportions relative to thewild-type strain taken as 100%. FIG. 50 represents the proportion ofeach component based on the total amount of the fatty acids, whichincludes AA: 1.5%, DGLA: 1.8%, ETA: 0.5%, EPA: 11.4%, n-6DPA: 1.2%, andDHA: 22.0%, which can also be described as the values of GC area such asn-6DPA/DTA: 0.6, DHA/n-3DPA: 5.7, C20PUFA/C22PUFA: 0.5, andn-6PUFA/n-3PUFA: 0.2.

As a result, the arachidonic acid was reduced to about 1/7, and the DPAto about 1/10. EPA and DHA increased by a factor of about 3.

Example 7

[Disruption of Thraustochytrium roseum C20 Elongase Gene]

[Example 7-1] Cloning of T. roseum-Derived C20 Elongase Gene

A forward denatured oligonucleotide (EL020F;5′-ATH GAR TWY TKB RTI TTYGTI CA-3′) (SEQ ID NO: 179) and a reverse denatured oligonucleotide(EL020R;5′-TAR TRI SWR TAC ATI ADI AMR TG-3′) (SEQ ID NO: 180) weresynthesized by targeting a conserved region in the C20 elongase gene ofthe Thraustochytrium roseum ATCC 28210 strain. Then, a PCR was performedwith an Advantage 2 Polymerase Mix (Clontech), using the T. roseumgenomic DNA extracted by using the same technique described in themethod of Example 2-5 as a template [PCR cycles: 94° C. 1 min/94° C. 30sec, 55° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. Theresulting specific product was isolated by 2% agarose gelelectrophoresis, and purified. The DNA fragment was then TA cloned witha pGEM-T easy Vector (Promega), and the base sequence was analyzed. Thesequence showed significant sequence identity with the sequence of aknown T. aureum-derived C20 elongase gene, suggesting that the sequencewas a partial sequence of the T. roseum-derived C20 elongase gene.

This was followed by cloning of the T. roseum-derived C20 elongase geneby 3′- and 5′-RACE, as in Example 2-2. First, the followingoligonucleotide primers were designed.

Forward oligonucleotide primer (8 F1; 5′-CTG ACA AAG TTT CTC GAC TGG AGCGAC A-3′) (SEQ ID NO: 181)

Reverse oligonucleotide primers (8 R1; 5′-TAC GCG GCG GTG CCC GAG CCCCAG-3′) (SEQ ID NO: 182) and (8 R2; 5′-TGC CGA TCG TTG CGT GGT GGA ACACCT G-3′) (SEQ ID NO: 183)

This was followed by 3′- and 5′-RACE using a synthetic adapter-specificoligonucleotide, and the oligonucleotide 8 F1 or 8 R1, using the cDNAlibrary created with a SMART™ RACE cDNA Amplification Kit (clontech) asa template [PCR cycles: 94° C. 30 sec 5 cycles/94° C. 30 sec, 70° C. 30sec, 72° C. 3 min, 5 cycles/94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min,25 cycles/4° C. ∞]. In the 5′RACE, a nested PCR was performed by using asynthetic adapter-specific oligonucleotide and the oligonucleotide 8 R2,using the RACE product as a template [PCR cycles: 94° C. 1 min/94° C. 30sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/72° C. 10 min/4° C. ∞]. Theboth specific products were gel purified, and the base sequence wasanalyzed after being TA cloned with a pGEM-T easy Vector (Promega).There was a complete match with the T. aureum. ATCC 34304-derived C20elongase (TaELO2) (SEQ ID NO: 16) of Example 2-2.

Then, a forward oligonucleotide (8 ORF F; 5′-ATG GCG ACG CGC ACC TCGAA-3′) (SEQ ID NO: 184) and a reverse oligonucleotide (8 ORF R; 5′-TTACTC GGA CTT GGT GGG GGC G-3′) (SEQ ID NO: 185) for amplifying a putativetranslated sequence were synthesized, and a PCR was performed with anAdvantage GC 2 polymerase Mix (Clontech), using the T. roseum genomicDNA as a template [PCR cycles: 94° C. 1 min/94° C. 30 sec, 65° C. 30sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. The resultingspecific product was gel purified, and the base sequence was analyzed bydirect sequencing. The T. roseum-derived C20 elongase gene was found tobe identical to the TaELO2. As demonstrated above, the sequence had acomplete match with the sequence of the Thraustochytrium aureum C20elongase. The base sequence is represented by SEQ ID NO: 186, and theamino acid sequence by SEQ ID NO: 187.

[Example 7-2] Production of Base Plasmid for C20 Elongase Gene TargetingVector Production

The Thraustochytrium aureum ATCC 34304 strain was cultured in a GYmedium. Cells at the late stage of the logarithmic growth phase werecentrifuged at 4° C., 3,500×g for 5 min to form a pellet, and disruptedafter being frozen with liquid nitrogen. After being extracted withphenol, the cell disruption liquid was precipitated with ethanol, andthe precipitate was dissolved in a TE solution. The nucleic acidsdissolved in the TE solution were treated with RNase at 37° C. for 30min to degrade the RNA. After being reextracted with phenol, the productwas precipitated with ethanol, and the precipitate was dissolved in a TEsolution. The DNA concentration was calculated by measuring A260/280. Byusing this as a template, the sequence (3,193 bp, SEQ ID NO: 188)containing the C20 elongase gene sequence was amplified with an LA taqHot start version (Takara Bio) [PCR cycles: 98° C. 2 min/98° C. 10 sec,68° C. 1 min, 30 cycles/68° C. 2 min]. The E2 KO ProF EcoRV (SEQ ID NO:33) and E2KO TermR EcoRV (SEQ ID NO: 34) of Example 2-8 were used as PCRprimers. The resulting DNA fragment was cloned into a pGEM-T easy vector(Promega), amplified with Escherichia coli, and the sequence wasconfirmed by using a Dye Terminator Cycle Sequencing Kit (BECKMANCOULTER). This was named pRH59 (FIG. 51).

By using the pRH59 (FIG. 51) as a template, amplification was performedwith a PrimeSTAR Max DNA Polymerase (Takara Bio) using a primer set ofthe reverse orientation prepared for the insertion of the BglII site ina portion halfway along the C20 elongase gene sequence. The primers usedare as follows. The both primers have BglII linker sequences [RHO120: 27mer: 5′-GAC AAA GAT CTC GAC TGG AGC GAC CAC-3′ (SEQ ID NO: 189), RHO121:27 mer: 5′-GTC GAG ATC TTT TGT CAG GAG GTG CAC-3′ (SEQ ID NO: 190)] [PCRcycles: 98° C. 2 min/98° C. 10 sec, 56° C. 15 sec, 72° C. 1 min, 30cycles/72° C. 1 min]. After the amplification performed under theseconditions, the product was digested with BglII, and allowed to selfligate. The ligated sample was amplified with Escherichia coli, and thesequence was confirmed by using a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER). This was named pRH64. The C20 elongase gene sequence951 bp with the inserted BglII site is represented by SEQ ID NO: 191.

The produced base plasmid (pRH64) for the production of theThraustochytrium roseum C20 elongase gene targeting vector is shown inFIG. 52.

[Example 7-3] Production of Targeting Vectors (ArtificialNeomycin-Resistant Gene and Hygromycin-Resistant Gene)

The pRH31 (FIG. 13) of Example 3-2 was digested with BglII, and the DNAfragment containing an artificial neomycin-resistant gene cassette wasligated to the BglII site of the pRH64 (FIG. 52) of Example 7-2. Thiswas named pRH65.

The pRH32 (FIG. 15) of Example 3-3 was digested with BglII, and the DNAfragment containing a hygromycin-resistant gene cassette was ligated tothe BglII site of the pRH64 (FIG. 52) of Example 7-2. This was namedpRH66.

The two targeting vectors (pRH65 and 66) produced are shown in FIG. 53.

[Example 7-4] Introduction of C20 Elongase Gene Targeting Vector

By using the two targeting vectors produced in Example 7-3 as templates,the gene was amplified with a PrimeSTAR GXL polymerase (Takara Bio),using a forward primer containing a translation initiation site (RHO130:5′-ATG GCG ACG CGC ACC TCG AAG AG-3′) (SEQ ID NO: 192) and a reverseprimer containing a translation termination site (RHO131: 5′-TTA CTC GGACTT GCT GGG GGC GC) (SEQ ID NO: 193) as primers [PCR cycles: 98° C. 2min/98° C. 30 sec, 60 30 sec, 72° C. 3 min, 30 cycles]. After beingextracted with phenol-chloroform and then with chloroform, the DNA wasprecipitated with ethanol, and the precipitate was dissolved in 0.1×TE.The DNA concentration was calculated by measuring A260/280. Theintroduced fragment obtained from using the pRH65 (FIG. 53) of Example7-3 as a template was 2,655 bp, and had the following sequence order:First half of Thraustochytrium aureum C20 elongase gene-SV40 terminatorsequence-artificial neomycin-resistant gene sequence-ubiquitin promotersequence-second half of Thraustochytrium aureum C20 elongase gene (SEQID NO: 194). The introduced fragment obtained from using the pRH66 (FIG.53) of Example 7-3 as a template was 2,887 bp, and had the followingsequence order: First half of Thraustochytrium aureum C20 elongasegene-ubiquitin promoter sequence-hygromycin-resistant gene sequence-SV40terminator sequence-second half of Thraustochytrium aureum C20 elongasegene (SEQ ID NO: 195).

The Thraustochytrium roseum strain was cultured in a GY medium for 7days, and cells in the logarithmic growth phase were used for geneintroduction. The DNA fragment (0.625 μg) was then introduced into cellscorresponding to OD600=1 to 1.5 using the gene-gun technique under thefollowing conditions (microcarrier: 0.6-micron gold particles, targetdistance: 6 cm, chamber vacuum: 26 mmHg, rupture disk: 900 PSI). After a24-hour recovery time, the cells with the introduced gene were appliedto a PDA plate medium (containing 2 mg/ml G418 or 2 mg/ml hygromycin).

As a result, about 20 drug resistant strains were obtained perpenetration.

[Example 7-5] Identification of C20 Elongase Gene Gene TargetingHomologous Recombinant

The Thraustochytrium roseum ATCC 28210 strain, the C20 elongase genehetero homologous recombinant, and the C20 elongase gene homo homologousrecombinant (gene disrupted strain) were cultured in GY media. Theresulting cells were centrifuged at 4° C., 3,000 rpm for 10 min to formapellet, and lysed at 55° C., 6 h/99.9° C., 5 min after being suspendedin a 20-μ1 SNET solution [20 mM Tris-HCl; pH 8.0, 5 mM NaCl, 0.3% SDS,200 μg/ml Proteinase K (nacalaitesque)]. The resulting cell lysate wasdiluted 10 times and used as a template in a PCR performed with a MightyAmp DNA polymerase (Takara Bio) to confirm the genome structure. Thepositions of the primers, combinations used for the amplification, andthe expected sizes of the amplification products are shown in FIG. 54.RoseumF and RoseumR were set upstream and downstream of the C20elongase, respectively. NeoF and NeoR were set on the artificialneomycin-resistant gene. HygF and HygR were set on thehygromycin-resistant gene [RoseumF: 26 mer: 5′-GCT CGG CTG GAA GTT GAGTAG TTT GC-3′ (SEQ ID NO: 196), RoseumR: 24 mer: 5′-TCT TTC TTC GTC GACGTC CCA CTG-3′ (SEQ ID NO: 197), NeoF: 24 mer: 5′-ATG ATT GAA CAG GACGGC CTT CAC-3′ (SEQ ID NO: 198), NeoR: 24 mer: 5′-TCA AAA GAA CTC GTCCAG GAG GCG-3′ (SEQ ID NO: 199), HygF: 24 mer: 5′-ATG AAA AAG CCT GAACTC ACC GCG-3′ (SEQ ID NO: 200), HygR: 25 mer: 5′-CTA TTC CTT TGC CCTCGG ACG AGT G-3′ (SEQ ID NO: 201)] [PCR cycles: 98° C. 2 min/98° C. 10sec, 60° C. 15 sec, 68° C. 4 min, 30 cycles].

C20 elongase knockout strains were obtained that showed no amplificationof the wild-type allele (Wt allele) but showed amplification of theartificial neomycin-resistant gene allele (NeoR allele) andhygromycin-resistant gene allele (HygR allele) (FIG. 55).

[Example 7-6] Changes in Fatty Acid Composition by C20 ElongaseDisruption

The Thraustochytrium roseum ATCC 28210 strain and the gene disruptedstrain were cultured in GY media. Cells at the late stage of thelogarithmic growth phase were centrifuged at 4° C., 3,000 rpm for 10 minto form a pellet, suspended in 0.9% NaCl, and washed. The cells werefurther centrifuged at 4° C., 3,000 rpm for 10 min, and the pellet wassuspended in sterile water, and washed. This was centrifuged at 3,000rpm for 10 min, and freeze dried after removing the supernatant. Then, 2ml-methanolic KOH (7.5% KOH in 95% methanol) was added to the freezedried cells, and, after being vortexed, the cells were ultrasonicallydisrupted (80° C., 30 min). The cells were vortexed after adding sterilewater (500 μl), and vortexed again after adding n-hexane (2 ml). Thiswas followed by centrifugation at 3,000 rpm for 10 min, and the upperlayer was discarded. The cells were vortexed again after adding n-hexane(2 ml), and centrifuged at 3,000 rpm for 10 min. After discarding theupper layer, 6 N HCl (1 ml) was added to the remaining lower layer, andthe mixture was vortexed. The mixture was vortexed again after addingn-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10min, and the upper layer was collected. The mixture was further vortexedafter adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, andthe upper layer was collected. The collected upper layer was thenconcentrated and dried with nitrogen gas. The concentrated dry samplewas incubated overnight at 80° C. after adding 3 N methanolic HCl (2ml).

The sample was allowed to cool to room temperature, and 0.9% NaCl (1 ml)was added. The mixture was vortexed after adding n-hexane (2 ml). Thiswas followed by centrifugation at 3,000 rpm for 10 min, and the upperlayer was collected. The mixture was further vortexed after addingn-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upperlayer was collected. After adding a small amount of anhydrous sodiumsulfate to the collected upper layer, the mixture was vortexed, andcentrifuged at 3,000 rpm for 10 min. After collecting the upper layer,the upper layer was concentrated and dried with nitrogen gas. Theconcentrated dry sample was dissolved in n-hexane (0.2 ml), and 2 μl ofthe sample was GC analyzed. The GC analysis was performed with a gaschromatograph GC-2014 (Shimadzu Corporation) under the followingconditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

As a result, knocking out the C20 elongase in the Thraustochytriumroseum increased fatty acids of 20 carbon chain length (FIG. 56). FIG.57 represents the proportions relative to the wild-type strain taken as100%. FIG. 57 represents each proportion of the components based on thetotal amount of the fatty acids, which include AA: 5.5%, EPA: 8.5%,n-6DPA: 13.2%, and DHA: 43.9%.

As can be seen from these results, the arachidonic acid increased about1.2-fold, EPA about 1.6-fold, DPA about 1.2-fold, and DHA about1.5-fold.

Example 8

Disruption of 44 Desaturase Gene in Thraustochytrium aureum ATCC 34304OrfA Disrupted Strain

[Example 8-1] Cloning of Sequence from 1,071 bp Upstream of 44Desaturase Gene to 1,500 bp within 44 Desaturase Gene inThraustochytrium aureum ATCC 34304 Strain

The genomic DNA of the Thraustochytrium aureum ATCC 34304 strainextracted by using the method described in Example 3-2 was decoded.Then, a search was made for a gene sequence highly homologous to a known44 desaturase, and two PCR primers were designed by using the searchresult. TMO3 is a sequence located 1,071 to 1,049 bp upstream of the Δ4desaturase gene of the Thraustochytrium aureum ATCC 34304 strain. TMO4is a sequence within the protein coding region, located 1,477 to 1,500bp from the start codon [TMO3: 23 mer: 5′-GGC GGA GCG AAG TGT GAA AGTTA-3′ (SEQ ID NO: 202), TMO4: 24 mer: 5′-GCG ACA GCA TCT TGA AAT AGGCAG-3′ (SEQ ID NO: 203)]. By using the genomic DNA of theThraustochytrium aureum ATCC 34304 strain as a template, the sequencefrom 1,071 bp upstream of the Δ4 desaturase gene to 1,500 bp within theΔ4 desaturase gene of the Thraustochytrium aureum ATCC 34304 strain wasamplified with the two primers, using an LA taq Hot start version(Takara Bio). The amplification was performed under the followingconditions [PCR cycles: 98° C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72°C. 3 min, 30 cycles/72° C. 8 min]. The resulting DNA fragment was clonedinto a pGEM-T easy vector, amplified with Escherichia coli, and thesequence was confirmed by using a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER). This was named pTM1 (FIG. 58).

[Example 8-2] Production of Base Plasmid for 44 Desaturase GeneTargeting Vector Production

By using the pTM1 (FIG. 58) of Example 8-1 as a template, a primer setof the reverse orientation was prepared in a manner that allows the 60bp upstream of the Δ4 desaturase gene and a 556-bp sequence containingthe start codon within the Δ4 desaturase gene (616 bp, SEQ ID NO: 205)to be deleted, and a BglII site to occur in the deleted portion. TMO7and TMO8 both contain BglII sequences. A PrimeSTAR Max DNA Polymerase(Takara Bio) was used for the amplification [TMO7: 25 mer: 5′-CAG GAGATC TCC AAG TCG CGA TTC A-3′ (SEQ ID NO: 206), TMO8: 26 mer: 5′-CTT GGAGAT CTC CTG CCC GTC CCG AA-3′ (SEQ ID NO: 207)] [PCR cycles: 98° C. 3min/98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec, 30 cycles/72° C. 30sec]. After the amplification performed under these conditions, theproduct was electrophoresed on an agarose gel, and purified. Theresulting DNA fragment was introduced into Escherichia coli andamplified, and the sequence was confirmed by using a Dye TerminatorCycle Sequencing Kit (BECKMAN COULTER). This was named pTM2.

The product base plasmid (pTM2) for the Δ4 desaturase gene targetingvector production is shown in FIG. 59.

[Example 8-3] Production of Targeting Vectors (Blasticidin-ResistantGene and GFP-Fused Zeocin-Resistant Gene)

The pRH38 (FIG. 33) of Example 5-3 was digested with BglII, and the DNAfragment containing a blasticidin-resistant gene cassette was ligated tothe BglII site of the pTM2 (FIG. 59) of Example 8-2. This was namedpTM6.

The pRH51 (FIG. 36) of Example 5-4 was digested with BglII, and the DNAfragment containing a GFP-fused zeocin-resistant gene cassette wasligated to the BglII site of the pTM2 (FIG. 59) of Example 8-2. This wasnamed pTM8.

The two targeting vectors (pTM6 and 8) produced are shown in FIG. 60.

[Example 8-4] Introduction of 44 Desaturase Gene Targeting Vector intoThraustochytrium aureum OrfA Disrupted Strain

By using the two targeting vectors produced in Example 8-3 as templates,the gene was amplified with a PrimeSTAR HS DNA polymerase (Takara Bio),using TMO3 (Example 8-1; SEQ ID NO: 202) and TMO4 (Example 8-1; SEQ IDNO: 203) as primers [PCR cycles: 98° C. 3 min/98° C. 10 sec, 55° C. 5sec, 72° C. 4 min, 30 cycles/72° C. 3 min].

After being extracted with phenol-chloroform and then with chloroform,the DNA was precipitated with ethanol, and the precipitate was dissolvedin 0.1×TE. The DNA concentration was calculated by measuring A260/280.The introduced fragment obtained from using the pTM6 (FIG. 60) ofExample 8-3 as a template was 3,264 bp, and had the following sequenceorder: Upstream of Thraustochytrium aureum Δ4 desaturase gene-SV40terminator sequence-blasticidin-resistant gene sequence-ubiquitinpromoter-sequence within Thraustochytrium aureum Δ4 desaturase gene (SEQID NO: 208). The introduced fragment obtained from using the pTM8 (FIG.60) of Example 8-3 as a template was 3,935 bp, and had the followingsequence order: Upstream of Thraustochytrium aureum Δ4 desaturasegene-SV40 terminator sequence-zeocin-resistant gene sequence-enhancedGFP gene sequence-ubiquitin promoter-sequence within Thraustochytriumaureum Δ4 desaturase gene (SEQ ID NO: 209).

The gene disrupted strain of the PUFA PKS pathway-associated gene OrfAof Example 4 was cultured in a GY medium for 4 days, and cells in thelogarithmic growth phase were used for gene introduction. The DNAfragment (0.625 μg) was then introduced into cells corresponding toOD600=1 to 1.5 by using the gene-gun technique (microcarrier: 0.6-microngold particles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupturedisk: 1,100 PSI). After a 4- to 6-hour recovery time, the cells with theintroduced gene was applied to a PDA agar plate medium (containing 20mg/ml Zeocin or 0.2 mg/ml blasticidin). As a result, 100 to 200 drugresistant strains were obtained per penetration.

[Example 8-5] Identification of 44 Desaturase Gene Gene TargetingHomologous Recombinant

Genomic DNA was extracted from Thraustochytrium aureum, and the Δ4desaturase gene disrupted strain of the Thraustochytrium aureum OrfAdisrupted strain by using the method of Example 3-2.

The DNA concentration was calculated by measuring A260/280. By usingthis as a template, a PCR was performed with a Mighty Amp DNA polymerase(Takara Bio) to confirm the genome structure. The positions of theprimers, combinations used for the amplification, and the expected sizesof the amplification products are shown in FIG. 61. TMO1 was setupstream of the Δ4 desaturase gene. TMO2 was set downstream of the Δ4desaturase gene. RHO198 and RHO49 (Example 5-3; SEQ ID NO: 139) were seton the blasticidin-resistant gene. RHO128 was set on the enhanced GFPgene. RHO64 (Example 5-4; SEQ ID NO: 151) was set on thezeocin-resistant gene [TMO1: 23 mer: 5′-AAA AGA ACA AGC CCT CTC CTGGA-3′ (SEQ ID NO: 210), TMO2: 23 mer: 5′-GAG GTT TGT ATG TTC GGC GGTTT-3′ (SEQ ID NO: 211), RHO198: 26 mer: 5′-TGG GGG ACC TTG TGC AGA ACTCGT GG-3′ (SEQ ID NO: 212), RHO128: 22 mer: 5′-GAC CTA CGG CGT GCA GTGCTT C-3′ (SEQ ID NO: 213)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68°C. 4 min 30 sec, 30 cycles/68° C. 4 min].

Δ4 desaturase gene knockout strains were obtained that showed noamplification of the wild-type allele (Wt allele) but showedamplification of the blasticidin-resistant gene allele (BlaR allele) andzeocin-resistant gene allele (ZeoR allele) (FIG. 62). It was found bythe experiment that the Thraustochytrium aureum ATCC 34304 strain didnot become auxotrophic even with the deletion of the PKSpathway-associated gene OrfA and the Δ4 desaturase gene.

[Example 8-6] Changes in Fatty Acid Composition by Disruption of 44Desaturase Gene in Thraustochytrium aureum OrfA Disrupted Strain

The Thraustochytrium aureum ATCC 34304 and the gene disrupted strainwere cultured by using the method of Example 3-9. After freeze drying,the fatty acids were methylesterificated, and GC analyzed. The GCanalysis was performed with a gas chromatograph GC-2014 (ShimadzuCorporation) under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

Changes in fatty acid composition are represented in FIG. 63. FIG. 64represents the proportions relative to the wild-type strain taken as100%. FIG. 64 represents the proportion of each component based on thetotal amount of the fatty acids, which includes AA: 8.4%, DGLA: 0.8%,ETA: 0.3%, EPA: 6.2%, n-6DPA: 0.2%, and DHA: 0.5%, which can also bedescribed as the values of GC area such as n-6DPA/DTA: 0.02, DHA/n-3DPA:0.03, C20PUFA/C22PUFA: 0.6, and n-6PUFA/n-3PUFA: 0.9.

As can be seen from the results, disrupting the Δ4 desaturase gene inthe Thraustochytrium aureum OrfA disrupted strain resulted in hardlypeforming C22:5n-6 (DPA) and C22:6n-3 (DHA) biosyntheses, and C22:4n-6(DTA) and C22:5n-3 (DPA) accumulated.

Example 9

Disruption of C20 Elongase Gene in Parietichytrium sp. SEK358 Strain[Example 9-1] Introduction of C20 Elongase Gene Targeting Vector intoParietichytrium sp. SEK358 Strain

By using the targeting vector produced with the pRH85 (FIG. 18) ofExample 3-6 as a template, the gene was amplified with a PrimeSTAR MaxDNA polymerase (Takara Bio), using RHO153 (Example 3-4; SEQ ID NO: 74)and RHO154 (Example 3-4; SEQ ID NO: 75) as primers [PCR cycles: 98° C. 2min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min]. After beingextracted with phenol-chloroform and then with chloroform, the DNA wasprecipitated with ethanol, and the precipitate was dissolved in 0.1×TE.The DNA concentration was calculated by measuring A260/280. Theintroduced fragment obtained from using the pRH85 (FIG. 18) of Example3-6 as a template was 2,661 bp, and had the following sequence order:First half of Parietichytrium C20 elongase gene-SV40 terminatorsequence-artificial neomycin-resistant gene sequence-ubiquitin promotersequence-second half of Parietichytrium C20 elongase gene (Example 3-7;SEQ ID NO: 81). The Parietichytrium sp. SEK358 strain was cultured in aGY medium for 3 days, and cells in the logarithmic growth phase wereused for gene introduction. The DNA fragment (0.625 μg) was thenintroduced into cells corresponding to OD600=1 to 1.5 using the gene-guntechnique (microcarrier: 0.6-micron gold particles, target distance: 6cm, chamber vacuum: 26 mmHg, rupture disk: 900 PSI). After a 24-hourrecovery time, the cells with the introduced gene were applied to a PDAagar plate medium containing 0.5 mg/ml G418. As a result, 10 to 30 drugresistant strains were obtained per penetration.

[Example 9-2] Identification of C20 Elongase Gene Gene TargetingHomologous Recombinant

Genomic DNA was extracted from the Parietichytrium sp. SEK358 strain andthe C20 elongase gene disrupted strain by using the method of Example3-2. The DNA concentration was calculated by measuring A260/280. Byusing this as a template, a PCR was performed with a Mighty Amp DNApolymerase (Takara Bio) to confirm the genome structure. The positionsof the primers, combinations used for the amplification, and theexpected sizes of the amplification products are as described in Example3-8 (FIG. 19).

RHO184 (Example 3-8; SEQ ID NO: 87) was set upstream of the C20elongase. RHO185 (Example 3-8; SEQ ID NO: 88) was set downstream of theC20 elongase. RHO142 (Example 3-8; SEQ ID NO: 85) and RHO143 (Example3-8; SEQ ID NO: 86) were set on the artificial neomycin-resistant gene[PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 2 min, 30 cycles/68° C.7 min].

C20 elongase knockout strains were obtained that showed no amplificationof the wild-type allele (Wt allele), but showed amplification of theartificial neomycin-resistant gene allele (NeoR allele) (FIG. 65).

[Example 9-3] Changes in Fatty Acid Composition by Disruption of C20Elongase

The Parietichytrium sp. SEK358 strain and the gene disrupted strain werecultured by using the method of Example 3-9. After freeze drying, thefatty acids were methylesterificated, and GC analyzed. The GC analysiswas performed with a gas chromatograph GC-2014 (Shimadzu Corporation)under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

Changes in fatty acid composition are represented in FIG. 66. FIG. 67represents the proportions relative to the wild-type strain taken as100%. FIG. 67 represents the proportion of each component based on thetotal amount of the fatty acids, which includes AA: 21.4%, DGLA: 8.6%,ETA: 2.1%, EPA: 23.8%, n-6DPA: 0.5%, DHA: 0.9%, which can also bedescribed as the values of GC area such as n-6DPA/DTA: 1.8, DHA/n-3DPA:4.1, C20PUFA/C22PUFA: 29.6, and n-6PUFA/n-3PUFA: 1.1. As can be seenfrom the results, knocking out the C20 elongase in the Parietichytriumsp. SEK358 strain caused reduction of fatty acids of 22 or greatercarbon chain length, and increased fatty acids of 20 carbon chainlength. Specifically, the arachidonic acid increased about seven-fold,and the EPA about eleven-fold. The DPA and DHA reduced to about 1/15 andabout ⅛, respectively.

Example 10

Disruption of C20 Elongase Gene in Parietichytrium sp. SEK571 Strain

[Example 10-1] Introduction of C20 Elongase Gene Targeting Vector intoParietichytrium sp. SEK571 Strain

By using the targeting vector produced with the pRH85 (FIG. 18) ofExample 3-6 as a template, the gene was amplified with a PrimeSTAR MaxDNA polymerase (Takara Bio), using RHO153 (Example 3-4; SEQ ID NO: 74)and RHO154 (Example 3-4; SEQ ID NO: 75) as primers [PCR cycles: 98° C. 2min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min]. After beingextracted with phenol-chloroform and then with chloroform, the DNA wasprecipitated with ethanol, and the precipitate was dissolved in 0.1×TE.The DNA concentration was calculated by measuring A260/280. Theintroduced fragment obtained from using the pRH85 (FIG. 18) of Example3-6 as a template was 2,661 bp, and had the following sequence order:First half of Parietichytrium C20 elongase gene-SV40 terminatorsequence-artificial neomycin-resistant gene sequence-ubiquitin promotersequence-second half of Parietichytrium C20 elongase gene (Example 3-7;SEQ ID NO: 81). The Parietichytrium sp. SEK571 strain was cultured in aGY medium for 3 days, and cells in the logarithmic growth phase wereused for gene introduction. The DNA fragment (0.625 μg) was thenintroduced into cells corresponding to OD600=1 to 1.5 using the gene-guntechnique (microcarrier: 0.6-micron gold particles, target distance: 6cm, chamber vacuum: 26 mmHg, rupture disk: 1550 PSI). After a 24-hourrecovery time, the cells with the introduced gene were applied to a PDAagar plate medium containing 0.5 mg/ml G418. As a result, 5 to 15 drugresistant strains were obtained per penetration.

[Example 10-2] Identification of C20 Elongase Gene Gene TargetingHomologous Recombinant

Genomic DNA was extracted from the Parietichytrium sp. SEK571 strain andthe C20 elongase gene disrupted strain by using the method of Example3-2, and the DNA concentration was calculated by measuring A260/280. Byusing this as a template, a PCR was performed with a Mighty Amp DNApolymerase (Takara Bio) to confirm the genome structure. The positionsof the primers, combinations used for the amplification, and theexpected sizes of the amplification products are as described in Example3-8 (FIG. 19).

RHO184 (Example 3-8; SEQ ID NO: 87) was set upstream of the C20elongase. RHO185 (Example 3-8; SEQ ID NO: 88) was set downstream of theC20 elongase. RHO142 (Example 3-8; SEQ ID NO: 85) and RHO143 (Example3-8; SEQ ID NO: 86) were set on the artificial neomycin-resistant gene[PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 2 min, 30 cycles/68° C.7 min].

C20 elongase knockout strains were obtained that showed no amplificationof the wild-type allele (Wt allele), but showed amplification of theartificial neomycin-resistant gene allele (NeoR allele) (FIG. 68).

[Example 10-3] Changes in Fatty Acid Composition by C20 ElongaseDisruption

The Parietichytrium sp. SEK571 strain and the gene disrupted strain werecultured by using the method of Example 3-9. After freeze drying, thefatty acids were methylesterificated, and GC analyzed. The GC analysiswas performed with a gas chromatograph GC-2014 (Shimadzu Corporation)under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

Changes in fatty acid composition are represented in FIG. 69. FIG. 70represents the proportions relative to the wild-type strain taken as100%. FIG. 70 represents the proportion of each component based on thetotal amount of the fatty acids, which includes AA: 13.2%, DGLA: 1.9%,ETA: 1.1%, EPA: 29.6%, n6DPA: 1.0%, and DHA: 1.2%, which can also bedescribed as the values of GC area such as n-6DPA/DTA: 6.4, DHA/n-3DPA:4.7, C20PUFA/C22PUFA: 14.6, and n-6PUFA/n-3PUFA: 0.7. As can be seenfrom the results, knocking out the C20 elongase in the Parietichytriumsp. SEK571 strain caused reduction of fatty acids of 22 or greatercarbon chain length, and increased fatty acids of 20 carbon chainlength. Specifically, the arachidonic acid increased about four-fold,and the EPA about eight-fold. The DPA and DHA both reduced to about1/12.

Example 11

Disruption of Thraustochytrium aureum ATCC 34304-Derived 412 DesaturaseGene

[Example 11-1] Isolation of Thraustochytrium aureum ATCC 34304-Derived412 Desaturase Gene

By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as atemplate, a Thraustochytrium aureum ATCC 34304-derived Δ12 desaturasegene was amplified by a PCR performed with a forward oligonucleotideprimer Tω3-F1 (22 mer: 5′-ATG TGC AAG GTC GAT GGG ACA A-3′) (SEQ ID NO:214) and a reverse oligonucleotide primer Tω3-R1 (22 mer: 5′-TCA CAA ACATCG CAG CCT TCG G-3′) (SEQ ID NO: 215) (enzyme used: LA taq Hot StartVersion, TaKaRa; PCR cycles: 98° C. 2 min/98° C. 30 sec, 53° C. 30 sec,72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞). As a result, a novel genesequence having a 1,185-bp (SEQ ID NO: 217) ORF, encoding 395 aminoacids (SEQ ID NO: 216) was obtained. In the amino acid sequence of thegene, three histidine boxes commonly conserved in desaturases, believedto construct the active site were conserved (FIG. 71). Further, becausethe gene showed high identity (41%, 44%, 41%) at the amino acid levelwith the Thalassiosira pseudonana-, Micromonas sp.-, and Phaeodactylumtricornutum-derived Δ12 desaturases in a Blast search (FIG. 71), it wasstrongly suggested that the gene was a Thraustochytrium aureum ATCC34304-derived Δ12 desaturase gene. In the following, the gene will bereferred to as TΔ12d.

[Example 11-2] Expression of TΔ12d using Budding Yeast Saccharomycescerevisiae as Host, and Analysis of Fatty Acid Composition of GeneIntroduced Strain

By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as atemplate, a DNA fragment containing HindIII and Xba I sites added to theboth ends of TΔ12d was prepared in a PCR performed with a forwardoligonucleotide primer Tω3-Hind3-F (30 mer: 5′-GGA AGC TTA TGT GCA AGGTCG ATG GGA CAA-3′) (SEQ ID NO: 218) and a reverse oligonucleotideprimer Tω3-XbaI-R (29 mer: 5′-TTC TAG ACT AGA GCT TTT TGG CCG CAC GC-3′)(SEQ ID NO: 219) (enzyme used: LA taq Hot Start Version, TaKaRa; PCRcycles: 98° C. 2 min/98° C. 30 sec, 53° C. 30 sec, 72° C. 1 min, 30cycles/72° C. 7 min/4° C. ∞). The DNA fragment was then incorporated inthe HindIII/Xba I site of a pYES2/CT vector to construct a TΔ12dexpression vector pYESTD12. The pYESTD12 and pYES2/CT were thenintroduced into yeasts by using the lithium acetate method. In the GCanalysis of the fatty acid composition of the TΔ12d overexpressingstrain (pYESTD12 introduced strain), novel peaks were confirmed atpositions corresponding to the retention times of LA (C18:249,12) andC16:249,12, but not in the mock introduced strain (pYES2/CT introducedstrain). FIG. 72 represents a GC analysis chart, and fatty acid levelsper dry cell. On the other hand, no conversion activity for other fattyacids [LA, GLA (C18:346,9,12), C20:2411,14, DGLA (C20:348,11,14), ARA(C20:445,8,11,14), DTA (C22:447,10,13,16)] was confirmed in the TΔ12doverexpressing strain. It became clear from these results that the TΔ12dwas a Thraustochytrium aureum ATCC 34304-derived 412 desaturase gene.

[Example 11-3] Construction of TΔ12d Targeting Vector

By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as atemplate, the upstream and downstream sequences (1,001 bp each) of theTΔ12d ORF were amplified in a PCR performed under the followingconditions (enzyme used: PrimeSTAR GXL, TaKaRa); PCR cycles: 98° C. 2min/98° C. 30 sec, 53° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7min/4° C. ∞). The following forward and reverse oligonucleotide primerswere used.

TD12d-up-F (SEQ ID NO: 220)(23 mer: 5′-AGT CAG CCC AGG CAC CGA TGA CG-3′) and TD12d-up-R(SEQ ID NO: 221) (39 mer: 5′-AGC CAG AGC TAG ATC TCT TGT GCT CCTTTT CAA TCC TTT-3′) TD12d-down-F (SEQ ID NO: 222)(39 mer: 5′-GGA GCA CAA GAG ATC TAG CTC TGG CTC AAG GGA CAC CGT-3′) andTD12d-down-R (SEQ ID NO: 223)(24 mer: 5′-CAC AGA AAC TGC CTT CAC GGG TCT-3′)

The resulting both DNA fragments were joined by fusion PCR with a Bgl IIsite inserted therebetween, and incorporated in a pGEM-T easy Vector(Promega). Then, the hygromycin-resistant gene cassette of Example 3-3,and the blasticidin-resistant gene cassette of Example 5-3 wereincorporated at the Bgl II site of the resulting vector to constructTΔ12d KO targeting vectors. These were named pTD12dKOHyg andpTD12dKOBla. The construction scheme of the TΔ12d KO targeting vectorsare shown in FIG. 73.

[Example 11-4] Introduction of TΔ12d Targeting Vector toThraustochytrium aureum ATCC 34304, and Acquisition of TΔ12d DisruptedStrain

In order to obtain an efficient homologous recombinant by using a splitmarker method, two homologous recombination fragments were amplified bya PCR performed by using pTD12dKOHyg as a template [enzyme used: LA taqHot Start Version, TaKaRa; PCR cycles: 98° C. 2 min/98° C. 30 sec, 60°C. 30 sec, 72° C. X min (X=1 min/kbp), 30 cycles/72° C. 7 min/4° C. ∞](FIG. 74). The fragments were then introduced to the Thraustochytriumaureum ATCC 34304 by using the gene-gun technique. The following forwardand reverse oligonucleotide primers were used for the amplification ofthe homologous recombination fragments.

(SEQ ID NO: 220) TD12d-up-F and Hyg-Knock-R (SEQ ID NO: 224)(24 mer: 5′-TGT TAT GCG GCC ATT GTC CGT CAG-3′), and Hyg-Knock-F(SEQ ID NO: 225) (24 mer: 5′-TGC GAT CGC TGC GGC CGA TCT TAG-3′) and(SEQ ID NO: 223) TD12d-down-R

As a result, a homologous recombinant with the disrupted TΔ12d firstallele was obtained. Thereafter, by using pTD12dKOBla as a template, ahomologous recombination fragment for disrupting the second allele wasamplified by a PCR performed with the forward and reverseoligonucleotide primers TD12d-up-F (SEQ ID NO: 220) and TD12d-down-R(SEQ ID NO: 223) (enzyme used: LA taq Hot Start Version, TaKaRa) [PCRcycles: 98° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 3 min, 30cycles/72° C. 7 min/4° C. ∞]. The fragment was then introduced to thehomologous recombinant containing the disrupted first allele. Completedisruption of TΔ12d was verified by a PCR (using the genomic DNA belowas a template) and a RT-PCR performed for the detection ofhygromycin-resistant gene, blasticidin-resistant gene, and TΔ12d, or bysouthern blotting.

FIG. 75 represents the amplification results for thehygromycin-resistant gene, blasticidin-resistant gene, and TΔ12damplified by a PCR performed by using the genomic DNAs of the wild-typestrain, the TΔ12d first allele disrupted strain, and the TΔ12d disruptedstrain (two alleles are disrupted) as templates.

As a result, amplification of the hygromycin-resistant gene and theblasticidin-resistant gene contained in the introduced homologousrecombination fragment was confirmed in the TΔ12d disrupted strain.However, no amplification of the disrupted TΔ12d was confirmed. Thefollowing forward and reverse oligonucleotide primers were used for theamplification of the hygromycin-resistant gene, blasticidin-resistantgene, and TΔ12d.

Hyg-F (SEQ ID NO: 226) (26 mer: 5′-ATG AAA AAG CCT GAA CTC ACC GCGAC-3′) and Hyg-R (SEQ ID NO: 227)(25 mer: 5′-CTA TTC CTT TGC CCT CGG ACG AGT G-3′), Bla-F(SEQ ID NO: 228) (27 mer: 5′-ATG GCC AAG CCT TTG TCT CAA GAA GAA-3′),and Bla-R (SEQ ID NO: 229) (30 mer: 5′-TTA GCC CTC CCA CAC ATA ACC AGAGGG CAG-3′), (SEQ ID NO: 214) Tw3-F1, and (SEQ ID NO: 215) Tw3-R1

FIG. 76 represents the results of the mRNA detection performed by RT-PCRfor the hygromycin-resistant gene, blasticidin-resistant gene, and TΔ12din the wild-type strain, the TΔ12d first allele disrupted strain, andthe TΔ12d disrupted strain. As a result, mRNA was detected for thehygromycin-resistant gene and the blasticidin-resistant gene containedin the introduced homologous recombination fragment in the TΔ12ddisrupted strain. However, mRNA was not detected for the disruptedTΔ12d. Note that the primers used are the same primers as used for thePCR in which the genomic DNA was used as a template.

By using the genomic DNA of the Thraustochytrium aureum ATCC 34304 as atemplate, two DIG-labeled probes were prepared, and southern blottingwas performed with these probes. The following forward and reverseoligonucleotide primers were used for the preparation of the DIG-labeledprobes.

KO up-probe-F1 (SEQ ID NO: 230)(23 mer: 5′-GGG GTC GGC CGG TGC AGC CTT AG-3′) and KO up-probe-R1(SEQ ID NO: 231) (24 mer: 5′-GGC GGT CAG CGA TCG GTC GGA CTC-3′), andKO down-probe-F3 (SEQ ID NO: 232)(23 mer: 5′-GCT TGC GGC TCC TGT TGG GTG AC-3′) and KO down-probe-R3(SEQ ID NO: 233) (23 mer: 5′-ACG CCT GGC TGC CCA CCA TAA AC-3′)

As a result, the bands of the wild-type allele (upstream side 2,028 bp,downstream side 2,334 bp) disappeared in the TΔ12d disrupted strain, andbands of the homologous recombination fragments (upstream side 5,880 bpand 5,253 bp; downstream side 1,496 bp and 2,334 bp) containing thehygromycin-resistant gene and the blasticidin-resistant gene weredetected instead (FIG. 77).

The PCR using the genomic DNA as a template, the RT-PCR, and southernblotting made it clear that the TΔ12d was disrupted.

[Example 11-5] Phenotypic Analysis of TΔ12d Disrupted Strain

Cells cultured in a 250-ml GY liquid medium for 5 days were collected in10 ml portions, and absorbance at OD 600 nm was measured (n=3). Afterthe measurement, the cells were collected, and washed once withsterilized ultrapure water. After freeze drying, the dry cell weight wasmeasured after 1-hour drying with a desiccator (n=3). As a result, nosignificant difference was observed in the proliferation among thewild-type strain, the first allele disrupted strain, and the TΔ12ddisrupted strain (FIG. 78). The wild-type strain, the first alleledisrupted strain, and the TΔ12d disrupted strain were GC analyzed fortheir fatty acid compositions.

As a result, large fatty acid composition changes were observed.Accumulation of C18:1n9 (OA) in the TΔ12d disrupted strain wasparticularly prominent. FIG. 79 represents the proportion of eachcomponent in the fatty acid composition. FIG. 80 represents fatty acidlevels per milligram of dry cells.

Example 12

Disruption of C20 Elongase Gene and Expression of ω3 Desaturase Gene inThraustochytrium aureum ATCC 34304 OrfA Gene Disrupted Strain [Example12-1] Production of C20 Elongase Gene Targeting and Saprolegniadiclina-Derived ω3 Desaturase Expression Vector (Blasticidin-ResistantGene)

By using the pRH43 (FIG. 39) of Example 5-6 as a template, a primer setof the reverse orientation was prepared in a manner that allows the tworestriction enzyme KpnI sites to be deleted, and a BamHI site to occurin the deleted portion. RHO189 and RHO190 both contain BamHI sequences.A PrimeSTAR Max DNA Polymerase (Takara Bio) was used for theamplification [RHO189: 28 mer: 5′-TTA GCG GGA TCC CAA TTC GCC CTA TAGT-3′ (SEQ ID NO: 234), RHO190: 27 mer: 5′-AAT TGG GAT CCC GCT AAG TATCTC CCG-3′ (SEQ ID NO: 235)] [PCR cycles: 98° C. 2 min/98° C. 10 sec,55° C. 15 sec, 72° C. 40 sec, 31 cycles/72° C. 1 min]. After theamplification performed under these conditions, the product waselectrophoresed on an agarose gel, and purified. The resulting DNAfragment was introduced into Escherichia coli and amplified, and thesequence was confirmed by using a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER). This was named pRH101 (FIG. 81).

By using the pRH101 as a template, a primer set of the reverseorientation was prepared in a manner that allows for insertion of arestriction enzyme KpnI site. RHO191 and RHO192 both contain KpnIsequences. A PrimeSTAR Max DNA Polymerase (Takara Bio) was used for theamplification [RHO191: 28 mer: 5′-AGA TCT GGT ACC GCA GCG CCT GGT GCAC-3′ (SEQ ID NO: 236), RHO192: 27 mer: 5′-GCT GCG GTA CCA GAT CTG GTCGCG TTT-3′ (SEQ ID NO: 237)] [PCR cycles: 98° C. 2 min/98° C. 10 sec,55° C. 15 sec, 72° C. 40 sec, 31 cycles/72° C. 1 min]. After theamplification performed under these conditions, the product waselectrophoresed on an agarose gel, and purified. The resulting DNAfragment was introduced into Escherichia coli and amplified, and thesequence was confirmed by using a Dye Terminator Cycle Sequencing Kit(BECKMAN COULTER). This was named pRH102 (FIG. 82).

The pRH48 (FIG. 46) of Example 6-1 was digested with KpnI, and a DNAfragment containing a Saprolegnia diclina-derived ω3 desaturaseexpression cassette was ligated to the KpnI site of the pRH102 (FIG.82). This was named pRH103.

The product C20 elongase gene targeting and Saprolegnia diclina-derivedω3 desaturase expression vector pRH103 is shown in FIG. 83.

[Example 12-2] Introduction of C20 Elongase Gene Targeting andSaprolegnia diclina-Derived ω3 Desaturase Expression Vector intoThraustochytrium aureum OrfA Disrupted Strain

By using the C20 elongase gene targeting vector pRH54 (FIG. 39) ofExample 5-6 as a template, the gene was amplified with a PrimeSTAR MaxDNA polymerase (Takara Bio) using KSO11 (Example 5-7; SEQ ID NO: 159)and KSO12 (Example 5-7; SEQ ID NO: 160) as primers [PCR cycles: 98° C. 2min/98° C. 30 sec, 68° C. 2 min, 30 cycles/68° C. 2 min]. After beingextracted with phenol-chloroform and then with chloroform, the DNA wasprecipitated with ethanol, and the precipitate was dissolved in 0.1×TE.The DNA concentration was calculated by measuring A260/280. Theintroduced fragment was 3,887 bp, and had the following sequence order:Upstream of Thraustochytrium aureum C20 elongase gene-ubiquitinpromoter-Enhanced GFP gene sequence-zeocin-resistant gene sequence-SV40terminator sequence-downstream of Thraustochytrium aureum C20 elongasegene (Example 5-7; SEQ ID NO: 162). The C20 elongase gene targeting andSaprolegnia diclina-derived ω3 desaturase expression vector pRH103 (FIG.83) of Example 12-1 was digested with a restriction enzyme BamHI. Afterbeing extracted with phenol-chloroform and then with chloroform, the DNAwas precipitated with ethanol, and the precipitate was dissolved in0.1×TE. The DNA concentration was calculated by measuring A260/280. Theintroduced fragment was 5,611 bp, and had the following sequence order:Upstream of Thraustochytrium aureum C20 elongase gene-ubiquitinpromoter-Saprolegnia diclina-derived ω3 desaturase genesequence-ubiquitin terminator-ubiquitin promoter-blasticidin-resistantgene sequence-SV40 terminator-downstream of Thraustochytrium aureum C20elongase gene (SEQ ID NO: 238).

The PUFA PKS pathway-associated gene OrfA gene disrupted strain ofExample 4 was cultured in a GY medium for 4 days, and cells in thelogarithmic growth phase were used for gene introduction. The DNAfragment (0.625 μg) was introduced into cells corresponding to OD600=1to 1.5 by using the gene-gun technique (microcarrier: 0.6-micron goldparticles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk:1,100 PSI). After a 4- to 6-hour recovery time, the cells with theintroduced gene were applied to a PDA agar plate medium (containing 20mg/ml Zeocin or 0.2 mg/ml blasticidin). As a result, 20 to 60 drugresistant strains were obtained.

[Example 12-3] Introduction of Homologous Recombinant Containing C20Elongase Gene Targeting and Saprolegnia diclina-Derived ω3 DesaturaseExpression Vector Inserted in Genome

Genomic DNA was extracted from the Thraustochytrium aureum PUFA PKSpathway-associated gene OrfA disrupted strain, the C20 elongase genefirst allele homologous recombinant of the Thraustochytrium aureum OrfAdisrupted strain, and the disrupted strain by using the method describedin Example 3-2. The DNA concentration was then calculated by measuringA260/280.

The genomic DNA was cut with restriction enzymes, and electrophoresed ona 0.7% SeaKem GTG agarose gel (Takara Bio) in about 2 to 3 μg per well.This was transferred to a nylon membrane, and hybridized at 51° C. for16 hours with probes produced with a DIG system (Roche Applied Science).RHO94 (Example 5-8; SEQ ID NO: 163) and RHO95 (Example 5-8; SEQ ID NO:164) were used for the production of the 5′-end probe. RHO96 (Example5-8; SEQ ID NO:165) and RHO97 (Example 5-8; SEQ ID NO: 166) were usedfor the production of the 3′-end probe. The amplification was performedunder the following conditions, and an LA taq Hot start version (TakaraBio) was used for the amplification [PCR cycles: 98° C. 2 min/98° C. 30sec, 58° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min]. Therestriction enzymes used, and the probe positions are as shown in FIG.84. Detection of the hybridized probes was made by using a chromogenicmethod (NBT/BCIP solution). Bands of the sizes expected from thehomologous recombination of the drug resistant genes were observed inthe analyses of both the 5′ end and the 3′ end (FIG. 85).

[Example 12-4] Disruption of C20 Elongase Gene in Thraustochytriumaureum OrfA. Disrupted Strain and Changes in Fatty Acid Composition bySaprolegnia diclina-Derived ω3 Desaturase Expression

The Thraustochytrium aureum ATCC 34304wild-type strain, and theSaprolegnia diclina-derived ω3 desaturase expressing strain with thedouble disruption of the PKS pathway (orfA gene) and the C20 elongasegene were cultured by using the method of Example 3-9. After freezedrying, the fatty acids were methylesterificated, and GC analyzed. TheGC analysis was performed with a gas chromatograph GC-2014 (ShimadzuCorporation) under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

Changes in fatty acid composition are represented in FIG. 86. FIG. 87represents the proportions relative to the wild-type strain taken as100%. FIG. 87 represents the proportion of each component based on thetotal amount of the fatty acids, which includes AA: 16.0%, DGLA: 2.2%,ETA: 0.3%, EPA: 21.6%, n-6DPA: 2.1%, and DHA: 2.2%, which can also bedescribed as the values of GC area such as n-6DPA/DTA: 9.5, DHA/n-3DPA:10.3, C20PUFA/C22PUFA: 8.5, and n-6PUFA/n-3PUFA: 0.8.

It was found as a result that disrupting the C20 elongase gene andexpressing the Saprolegnia diclina-derived ω3 desaturase in theThraustochytrium aureum OrfA disrupted strain increases the C20:4n-6(AA) about six-fold and the C20:5n3 (EPA) about ten-fold, and decreasesthe C22:6n-3 (DHA) to about 1/16.

Example 13

Expression of ω3 Desaturase Gene in Parietichytrium sp. SEK571 C20Elongase Gene Disrupted Strain

[Example 13-1] Production of Saprolegnia diclina-Derived ω3 DesaturaseExpression Plasmid using Hygromycin as Drug-Resistance Marker

For the production of a Saprolegnia diclina-derived ω3 desaturaseexpression plasmid using hygromycin as a drug-resistance marker, aplasmid pRH107 (FIG. 88) was used as the base plasmid after partiallymodifying the restriction enzyme site by subcloning the ParietichytriumC20 elongase upstream sequence (904 bp, SEQ ID NO: 239) andParietichytrium C20 elongase downstream sequence (721 bp, SEQ ID NO:240) into a pGEM-T easy vector. For reference, the total pRH107 sequenceis presented (4,592 bp, SEQ ID NO: 241). The sequence as the base of theexpression plasmid production is not actively used for the introductionof cells in this experiment, and as such it is not necessarily requiredto use pRH107 as the base vector in similar experiments. In conducting asimilar experiment, a cloning vector having a KpnI site and a BamHI sitein proximity can be used instead. Here, the sequence between the KpnIsite and the BamHI site should be as short as possible, because it isintroduced into cells as a linker between the ω3 desaturase geneexpression cassette and the drug resistant gene expression cassette. Inthis experiment example, the sequence corresponds to the ParietichytriumC20 elongase downstream sequence 37 bp (SEQ ID NO: 242).

The pRH48 (FIG. 46) of Example 6-1 was digested with KpnI, and the DNAfragment containing the Saprolegnia diclina-derived ω3 desaturase genecassette was ligated to the KpnI site of pRH107 (FIG. 88). This wasnamed pRH108 (FIG. 89).

The pRH32 (FIG. 15) of Example 3-3 was digested with BglII, and the DNAfragment containing the hygromycin-resistant gene cassette was ligatedto the BamHI site of pRH108 (FIG. 89). This was named pRH109 (FIG. 90).

[Example 13-2] Introduction of Saprolegnia diclina-Derived ω3 DesaturaseExpression Plasmid into Parietichytrium sp. SEK571 C20 Elongase GeneDisrupted Strain

By using the pRH109 (FIG. 90) produced in Example 13-1 as a template,the DNA was amplified with a PrimeSTAR Max DNA polymerase (Takara Bio),using TMO42 (Example 6-1, SEQ ID NO: 168) and RHO52 (Example 3-1, SEQ IDNO: 52) as primers [PCR cycles: 94° C. 30 sec, 72° C. 1 min, 5cycles/94° C. 30 sec, 70° C. 30 sec, 72° C. 1 min, 5 cycles/94° C. 30sec, 68° C. 30 sec, 72° C. 1 min, 25 cycles/72° C. 2 min]. Theamplification product was collected form a 1.0% agarose gel, andprecipitated with ethanol. The precipitate was then dissolved in 0.1×TE.The DNA concentration was calculated by measuring A260/280. Theintroduced fragment obtained by the PCR was 4,448 bp, and had thefollowing sequence order: Ubiquitin promoter-ω3 desaturasegene-ubiquitin terminator-ubiquitin promoter-hygromycin-resistant genesequence-SV40 terminator sequence (SEQ ID NO: 243).

The Parietichytrium sp. SEK571 C20 elongase gene disrupted strainproduced in Example 10 was cultured in a GY medium for 3 days, and cellsin the logarithmic growth phase were used for gene introduction. The DNAfragment (0.625 μg) was introduced into cells corresponding to OD600=1to 1.5 by using the gene-gun technique (microcarrier: 0.6-micron goldparticles, target distance: 6 cm, chamber vacuum: 26 mmHg, rupture disk:1550 PSI). After a 24-hour recovery time, the cells with the introducedgene were applied to a PDA agar plate medium (containing 1.0 mg/mlhygromycin). As a result, 5 to 20 drug resistant strains were obtainedper penetration.

[Example 13-3] Acquisition of Saprolegnia diclina-Derived ω3 DesaturaseGene Expressing Strain

Genomic DNA was extracted from the Parietichytrium sp. SEK571 C20elongase gene disrupted strain produced in Example 10 and the ω3desaturase gene expressing strain by using the method described inExample 3-2, and the DNA concentration was calculated by measuringA260/280. By using this as a template, a PCR was performed with an LAtaq Hot start version to confirm the genome structure. The positions ofthe primers, combinations used for the amplification, and the expectedsize of the amplification product are shown in FIG. 91. RHO90 (27 mer:5′-CGT TAG AAC GCG TAA TAC GAC TCA CTA-3′ SEQ ID NO: 244) was set on theubiquitin promoter, and RHO141 (Example 3-8, SEQ ID NO: 84) was set onthe hygromycin-resistant gene [PCR cycles: 98° C. 2 min/98° C. 10 sec,68° C. 4 min, 30 cycles/68° C. 7 min].

The result of amplification confirmed a band of the expected size (FIG.92). That is, a strain was isolated that contained the introducedexpression fragment stably introduced into its genome.

[Example 13-4] Changes in Fatty Acid Composition by ω3 DesaturaseExpression in Parietichytrium sp. SEK571 C20 Elongase Gene DisruptedStrain

The Parietichytrium sp. SEK571 strain, and the ω3 desaturase geneexpressing strain were cultured by using the method of Example 3-9.After freeze drying, the fatty acids were methylesterificated, and GCanalyzed. The GC analysis was performed with a gas chromatograph GC-2014(Shimadzu Corporation) under the following conditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

The ω3 desaturase expressing strain reduced levels of the n-6 seriesfatty acids, and there was a tendency for the n-3 series fatty acids toincrease (FIG. 93). FIG. 94 represents the proportions relative to thewild-type strain taken as 100%. FIG. 94 represents the proportion ofeach component based on the total amount of the fatty acids, whichincludes AA: 1.5%, DGLA: 0.2%, ETA: 0.5%, EPA: 5.2%, n-6DPA: 0.3%, andDHA: 0.4%, which can also be described as the values of GC area such asDHA/n-3DPA: 1.9, C20PUFA/C22PUFA: 8.2, and n-6PUFA/n-3PUFA: 0.3. As aresult, the arachidonic acid was reduced to about ½, and EPA increasedby a factor of about 1.4.

Example 14 Disruption of Schizochytrium C20 Elongase Gene [Example 14-1]Cloning of Schizochytrium-Derived C20 Elongase Gene

By using the genomic DNA extracted from Schizochytrium as a template, aSchizochytrium-derived C20 elongase gene was amplified by a PCRperformed with a forward oligonucleotide primer RHO134 (32 mer: 5′-CCCGGA TCC ATG GTG GCC AGC GAG GTG CTC AG-3′) (SEQ ID NO: 245) containing aBamHI site, and a reverse oligonucleotide primer RHO135 (34 mer: 5′-CCCGGA TCC TTA GTC GCG CTT GAG CTC AGC ATC C-3′) (SEQ ID NO: 246)containing a BamHI site (enzyme used: LA taq Hot Start Version, TaKaRa;PCR cycles: 98° C. 2 min/98° C. 30 sec, 53° C. 30 sec, 72° C. 1 min, 30cycles/72° C. 7 min/4° C. ∞). The both specific products were gelpurified, cloned into a pGEM-T easy vector (Promega), and amplified withEscherichia coli. The sequence was then confirmed by using a DyeTerminator Cycle Sequencing Kit (BECKMAN COULTER). This was named pRH70(FIG. 95). As a result of abase sequence analysis, a novel gene sequencehaving a 945-bp (SEQ ID NO: 248) ORF, encoding 315 amino acids (SEQ IDNO: 247) was obtained.

[Example 14-2] Production of Base Plasmid for ?C20 Elongase GeneTargeting Vector Production

By using the pRH70 (FIG. 95) produced in Example 14-1 as a template, thegene was amplified with a Prime STAR Max DNA Polymerase (Takara Bio),using a primer set of the reverse orientation prepared for insertion ofa BglII site in a portion halfway along the C20 elongase gene sequence.The primers used are as follows. The both had BglII linker sequences[RHO136: 25 mer: 5′-CAT CGA GAT CTT CGT GTT TGT CCA C-3′ (SEQ ID NO:249), RHO137: 25 mer: 5′-ACG AAG ATC TCG ATG CGG GCG TCC C-3′ (SEQ IDNO: 250)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 56° C. 15 sec, 72° C.1 min, 30 cycles/72° C. 1 min]. After the amplification performed underthese conditions, the product was digested with BglII, and allowed toself ligate. The ligated sample was amplified with Escherichia coli, andthe sequence was confirmed by using a Dye Terminator Cycle SequencingKit (BECKMAN COULTER). This was named pRH71. The C20 elongase genesequence 945 bp with the inserted BglII site is represented by SEQ IDNO: 251.

The product base plasmid (pRH71) for the production of theSchizochytrium C20 elongase gene targeting vector is shown in FIG. 96.

[Example 14-3] Production of Targeting Vectors (ArtificialNeomycin-Resistant Gene and Hygromycin-Resistant Gene)

The pRH31 (FIG. 13) of Example 2-2 was digested with BglII, and the DNAfragment containing an artificial neomycin-resistant gene cassette wasligated to the BglII site of the pRH71 (FIG. 96) of Example 14-2. Thiswas named pRH73.

The pRH32 (FIG. 15) of Example 2-3 was digested with BglII, and the DNAfragment containing a hygromycin-resistant gene cassette was ligated tothe BglII site of the pRH71 (FIG. 96) of Example 14-2. This was namedpKS-SKO.

The two targeting vectors (pRH73 and pKS-SKO) produced are shown in FIG.97.

[Example 14-4] Introduction of C20 Elongase Gene Targeting Vector

By using the two targeting vectors produced in Example 14-3 astemplates, the gene was amplified with a Prime STAR GXL polymerase,using a forward primer (SorfF: 20 mer: 5′-AGA TGG TGG CCA GCG AGG TG-3′)(SEQ ID NO: 252) containing a translation initiation site, and a reverseprimer (SorfR: 25 mer: 5′-TTA GTC GCG CTT GAG CTC AGC ATC C-3′) (SEQ IDNO: 253) containing a translation termination site [PCR cycles: 98° C. 2min/98° C. 30 sec, 60 30 sec, 72° C. 3 min, 30 cycles]. After beingextracted with phenol-chloroform and then with chloroform, the DNA wasprecipitated with ethanol, and the precipitate was dissolved in 0.1×TE.The DNA concentration was then calculated by measuring A260/280. Theintroduced fragment obtained from using the pRH73 (FIG. 97) of Example14-3 as a template was 2,644 bp, and had the following sequence order:First half of Schizochytrium C20 elongase gene-SV40 terminatorsequence-artificial neomycin-resistant gene sequence-ubiquitin promotersequence-second half of Schizochytrium C20 elongase gene (SEQ ID NO:254). The introduced fragment obtained from using the pKS-SKO (FIG. 97)of Example 14-3 as a template was 2,881 bp, and had the followingsequence order: First half of Schizochytrium C20 elongase gene-ubiquitinpromoter sequence-hygromycin-resistant gene sequence-SV40 terminatorsequence-second half of Schizochytrium C20 elongase gene (SEQ ID NO:255).

The Schizochytrium sp. TY12Ab strain was cultured in a GY medium for 7days, and cells in the logarithmic growth phase were used for geneintroduction. The DNA fragment (0.625 μg) was introduced into cellscorresponding to OD600=1 to 1.5 using the gene-gun technique(microcarrier: 0.6-micron gold particles, target distance: 6 cm, chambervacuum: 26 mmHg, rupture disk: 1,100 PSI). After a 24-hour recoverytime, the cells with the introduced gene were applied to a PDA platemedium (containing 2 mg/ml G418 or 2 mg/ml hygromycin).

As a result, about 20 drug resistant strains were obtained perpenetration.

[Example 14-5] Identification of C20 Elongase Gene Gene TargetingHomologous Recombinant

The Schizochytrium sp. TY12Ab strain (FERM BP-11421), the C20 elongasegene hetero homologous recombinant, and the C20 elongase gene homohomologous recombinant (gene disrupted strain) were cultured in GYmedia, and the resulting cells were centrifuged at 4° C., 3,000 rpm for10 min to form a pellet. The cells were then lysed at 55° C., 6 h/99.9°C., 5 min after being suspended in a 20-μ1 SNET solution [20 mMTris-HCl; pH 8.0, 5 mM NaCl, 0.3% SDS, 200 μg/ml Proteinase K (nacalaitesque)]. The resulting cell lysate was diluted 10 times and used as atemplate in a PCR performed with a Mighty Amp DNA polymerase (TakaraBio) to confirm the genome structure. The positions of the primers, andthe expected size of the amplification product are shown in FIG. 98. Theprimers were used in the SorfF and SorfR combination used in Example14-4 [PCR cycles: 98° C. 2 min/98° C. 10 sec, 60° C. 15 sec, 68° C. 4min, 30 cycles].

C20 elongase knockout strains were obtained that showed no amplificationof the wild-type allele (Wt allele), but showed amplification of theartificial neomycin-resistant gene allele (NeoR allele) andhygromycin-resistant gene allele (HygR allele) (FIG. 99).

[Example 14-6] Changes in Fatty Acid Composition by C20 ElongaseDisruption

The Schizochytrium sp. TY12Ab strain and the gene disrupted strain werecultured in GY media. Cells at the late stage of the logarithmic growthphase were centrifuged at 4° C., 3,000 rpm for 10 min to form a pellet,suspended in 0.9% NaCl, and washed. The cells were further centrifugedat 4° C., 3,000 rpm for 10 min, and the pellet was suspended in sterilewater, and washed. This was centrifuged at 3,000 rpm for 10 min, andfreeze dried after removing the supernatant. Then, 2 ml-methanolic KOH(7.5% KOH in 95% methanol) was added to the freeze dried cells, and,after being vortexed, the cells were ultrasonically disrupted (80° C.,30 min).

The cells were vortexed after adding sterile water (500 μl), andvortexed again after adding n-hexane (2 ml). This was followed bycentrifugation at 3,000 rpm for 10 min, and the upper layer wasdiscarded. The cells were vortexed again after adding n-hexane (2 ml),and centrifuged at 3,000 rpm for 10 min. After discarding the upperlayer, 6 N HCl (1 ml) was added to the remaining lower layer, and themixture was vortexed. The mixture was vortexed again after addingn-hexane (2 ml). This was followed by centrifugation at 3,000 rpm for 10min, and the upper layer was collected. The mixture was further vortexedafter adding n-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, andthe upper layer was collected. The collected upper layer was thenconcentrated and dried with nitrogen gas. The concentrated dry samplewas incubated overnight at 80° C. after adding 3 N methanolic HCl (2ml).

The sample was allowed to cool to room temperature, and 0.9% NaCl (1 ml)was added. The mixture was vortexed after adding n-hexane (2 ml). Thiswas followed by centrifugation at 3,000 rpm for 10 min, and the upperlayer was collected. The mixture was further vortexed after addingn-hexane (2 ml), centrifuged at 3,000 rpm for 10 min, and the upperlayer was collected. After adding a small amount of anhydrous sodiumsulfate to the collected upper layer, the mixture was vortexed, andcentrifuged at 3,000 rpm for 10 min. After collecting the upper layer,the upper layer was concentrated and dried with nitrogen gas. Theconcentrated dry sample was dissolved in n-hexane (0.2 ml), and 2 μl ofthe sample was GC analyzed. The GC analysis was performed with a gaschromatograph GC-2014 (Shimadzu Corporation) under the followingconditions:

Column: HR-SS-10 (30 m×0.25 mm; Shinwa Chemical Industries Ltd.)

Column temperature: 150° C.→(5° C./min)→220° C. (10 min)

Carrier gas: He (1.3 mL/min).

As a result, knocking out the C20 elongase in the Schizochytrium sp.TY12Ab strain increased fatty acids of 20 carbon chain length (FIG.100). FIG. 101 represents the proportions relative to the wild-typestrain taken as 100%. FIG. 101 represents the proportion of eachcomponent based on the total amount of the fatty acids, which includesAA: 17.2%, EPA: 3.3%, n-6DPA: 13.7%, and DHA: 15.6%, which can also bedescribed as the value of GC area such as DHA/n-3DPA: 38.8.

As can be seen from these results, the arachidonic acid increased about1.7-fold, EPA about 1.3-fold, DPA (n-6) about 1.1-fold, and DHA about0.9-fold. One of the EPA contents was increased to be 3.3% of a totalfatty acid composition. It has been found that the EPA content can be3.3% or more of a total fatty acid composition as shown by the aboveExamples.

INDUSTRIAL APPLICABILITY

The present invention provides a method for transforming stramenopilethrough disruption of stramenopile genes and/or inhibition of expressionthereof, modification of the fatty acid composition produced by astramenopile, and a method for highly accumulating fatty acids in astramenopile. The present invention thus enables more efficientproduction of polyunsaturated fatty acids.

What is claimed is:
 1. A method for producing a microbial oil,comprising: genetically modifying a labyrinthulid by disrupting and/orsilencing a gene, or by transforming another gene in addition to thedisruption and/or gene silencing of the gene; culturing thelabyrinthulid, such that a fatty acid composition accumulated in thelabyrinthulid comprises an increased EPA content; and collecting themicrobial oil having the increased EPA content from the labyrinthulid,wherein the increased EPA content is not less than 3.3% of a total fattyacid composition.
 2. The method for producing the microbial oilaccording to claim 1, wherein the disrupted and/or silenced gene is apolyketide synthase (PKS) gene, a fatty acid elongase gene and/or afatty acid desaturase gene, wherein the transformed another gene is afatty acid elongase gene and/or a fatty acid desaturase gene.
 3. Themethod for producing the microbial oil according to claim 2, wherein thepolyketide synthase (PKS) gene is OrfA, wherein the fatty acid elongasegene is a C20 elongase gene, and/or wherein the fatty acid desaturasegene is a Δ4 desaturase gene and/or an ω3 desaturase gene.
 4. The methodfor producing the microbial oil according to claim 1, wherein thegenetically modified labyrinthulid is able to grow in media which do notcontain PUFA.
 5. The method for producing the microbial oil according toclaim 1, wherein the step of disrupting or transforming the gene of alabyrinthulid utilizes electroporation or a gene gun method, and/orwherein the step of silencing the gene utilizes an antisense method orRNA interference.
 6. The method for producing the microbial oilaccording to claim 1, wherein the labyrinthulid belonging to the genusof Thraustochytrium, Parietichytrium, Schizochytrium, or Ulkenia.
 7. Themethod for producing the microbial oil according to claim 1, wherein thelabyrinthulid is Thraustochytrium aureum, Thraustochytrium roseum,Parietichytrium sarkarianum, Parietichytrium sp., or Schizochytrium sp.8. The method for producing the microbial oil according to claim 1,wherein the labyrinthulid is Thraustochytrium aureum (ATCC 34304),Thraustochytrium roseum (ATCC 28210), Parietichytrium sarkarianum SEK364(FERM BP-11298), Parietichytrium sp. SEK358 (FERM BP-11405),Parietichytrium sp. SEK571 (FERM BP-11406), or Schizochytrium sp. TY12Ab(FERM BP-11421).
 9. A labyrinthulid that has been genetically modifiedby disrupting and/or silencing a gene, or by transforming another genein addition to the disruption and/or gene silencing of the gene suchthat a fatty acid composition accumulated in the labyrinthulid comprisesan increased EPA content, wherein the increased EPA content is not lessthan 3.3% of a total fatty acid composition.
 10. The labyrinthulidaccording to claim 9, wherein the disrupted and/or silenced gene is apolyketide synthase (PKS) gene, a fatty acid elongase gene and/or afatty acid desaturase gene, and wherein the transformed another gene isa fatty acid elongase gene and/or a fatty acid desaturase gene.
 11. Thelabyrinthulid according to claim 10, wherein the polyketide synthase(PKS) gene is OrfA, wherein the fatty acid elongase gene is a C20elongase gene, and/or wherein the fatty acid desaturase gene is a Δ4desaturase gene and/or an ω3 desaturase gene.
 12. The labyrinthulidaccording to claim 9, wherein the labyrinthulid is able to grow in mediawhich do not contain PUFA.
 13. The labyrinthulid according to claim 9,wherein the labyrinthulid belongs to the genus of Thraustochytrium,Parietichytrium, Schizochytrium, or Ulkenia.
 14. The labyrinthulidaccording to claim 9, wherein the labyrinthulid is Thraustochytriumaureum, Thraustochytrium roseum, Parietichytrium sarkarianum,Parietichytrium sp., or Schizochytrium sp.
 15. The labyrinthulidaccording to claim 9, wherein the labyrinthulid is Thraustochytriumaureum (ATCC 34304), Thraustochytrium roseum (ATCC 28210),Parietichytrium sarkarianum SEK364 (FERM BP-11298), Parietichytrium sp.SEK358 (FERM BP-11405), Parietichytrium sp. SEK571 (FERM BP-11406), orSchizochytrium sp. TY12Ab (FERM BP-11421).